Neuropathy patterns differ in patients with diabetic complications.

PURPOSE: Diabetic polyneuropathy (PNP) is an important risk factor for foot ulcers. Diabetic dermopathy is more frequent in patients with diabetic neuropathy. We compared clinical and electrophysiological characteristics of PNP localizations/recurrences of foot ulcers, and diabetic dermopathy (DD) between sexes. METHODS: Eighty-eight diabetic patients (44 men, 44 women) had an evaluation regarding detailed history of their diseases, lesion-related data, and clinical examination. Nerve conduction velocities (NCV), compound motor action potentials (CMAP), distal latencies (DL), and sensory nerve action potentials were assessed from the right and left peroneal, right median/ulnar nerves. RESULTS: The presence of DD was more common in men (p < 0.001). The mean NCV of ulnar nerves was slower (p < 0.001); mean CMAP values were lower (p = 0.006); and mean DL was longer in men with compared to women (p = 0.003). Although EMG features of peroneal nerves showed no significant difference, diabetic men had more common and severe peroneal nerve involvement (p = 0.004). Carpal tunnel syndrome was more common in women, though not significant. Patients with right-sided ulcers had lower CMAP amplitudes on the right peroneal nerves in regard to left peroneal nerves (p = 0.009). CONCLUSIONS: Our findings suggest that ulnar nerves are more commonly involved in men, with lower CMAP slower NCV values, and longer DL values.

In Vivo Electrophysiological Measurement of the Rat Ulnar Nerve with Axonal Excitability Testing.

Electrophysiology enables the objective assessment of peripheral nerve function in vivo. Traditional nerve conduction measures such as amplitude and latency detect chronic axon loss and demyelination, respectively. Axonal excitability techniques "by threshold tracking" expand upon these measures by providing information regarding the activity of ion channels, pumps and exchangers that relate to acute function and may precede degenerative events. As such, the use of axonal excitability in animal models of neurological disorders may provide a useful in vivo measure to assess novel therapeutic interventions. Here we describe an experimental setup for multiple measures of motor axonal excitability techniques in the rat ulnar nerve. The animals are anesthetized with isoflurane and carefully monitored to ensure constant and adequate depth of anesthesia. Body temperature, respiration rate, heart rate and saturation of oxygen in the blood are continuously monitored. Axonal excitability studies are performed using percutaneous stimulation of the ulnar nerve and recording from the hypothenar muscles of the forelimb paw. With correct electrode placement, a clear compound muscle action potential that increases in amplitude with increasing stimulus intensity is recorded. An automated program is then utilized to deliver a series of electrical pulses which generate 5 specific excitability measures in the following sequence: stimulus response behavior, strength duration time constant, threshold electrotonus, current-threshold relationship and the recovery cycle. Data presented here indicate that these measures are repeatable and show similarity between left and right ulnar nerves when assessed on the same day. A limitation of these techniques in this setting is the effect of dose and time under anesthesia. Careful monitoring and recording of these variables should be undertaken for consideration at the time of analysis.

Fix the problem - A practical guide to whole-mount immunohistochemistry of teased nerve fibres.

BACKGROUND: Immunohistochemical staining of entire nerve fibres allows for studying the molecular composition of functional fibre subunits and may add to the diagnostic value of nerve fibre teasing. NEW METHOD: In this study, we established a sealed-slide method for reproducible immunostaining of deep axoplasmic proteins in permanently straightened nerve fibres. RESULTS: Immunostaining of teased nerve fibres very much is facilitated by tip-fixation with biocompatible glass adhesives. Antibody penetration in fresh nerves can be achieved by thermic and chemical permeabilisation while enzymatic digestion allows for sufficient permeability after aldehyde fixation. COMPARISON WITH EXISTING METHODS: The methods recommended herein are easy to perform and represent a reliable and reproducible way to whole mount immunostaining. CONCLUSIONS: Sealed-slide immunostaining of tip-fixed and permeabilised nerve biopsies will help to validate neurophysiological abnormalities and to screen for target molecules and predictive markers of peripheral nerve disorders such as in inherited neuropathies and Guillain-Barré syndrome.

Cross-chest median nerve transfer: a new model for the evaluation of nerve regeneration across a 40 mm gap in the rat.

A new animal model for the study of nerve regeneration in rats across a 40 mm gap between both median nerves is described. For autologous grafting, the ulnar nerves were dissected and sutured together. From the left median nerve, they were transplanted across the chest to the right median nerve. Animals having undergone this operation were observed for 12 months and periodically assessed using the grasping test and measurements of body-weight. For histological analysis rats were sacrificed after this period and axon counts were determined at the suture points of operated animals and in the median nerve of non-operated animals. Functional recovery could be seen, although partially, beginning as early as the fifth postoperative month, as demonstrated by the grasping test. Quantification of the number of axons demonstrated axonal regeneration across all three coaptation points. This model provides a new approach for analysis of long distance peripheral nerve regeneration without impairment of behaviour.

F wave study in amyotrophic lateral sclerosis: assessment of balance between upper and lower motor neuron involvement.

OBJECTIVE: We sought to record significant F wave variable changes in ALS patients having no advanced disease. Furthermore, an interpretation of these F wave abnormalities in the context of upper (UMN) and lower motor neuron (LMN) dysfunction was attempted. METHODS: Standard motor and sensory conduction study was performed to the ulnar nerves of 23 patients with ALS (13 males and 10 females with mean age 67.2+/-5.3 years), having a clinically predominant LMN syndrome. A series of 40 electrical stimuli were also delivered to both their ulnar nerves in order to obtain F waves. The following F wave variables were estimated: F persistence, F wave latency, amplitude, duration and F chronodispersion. Twenty-three, age-and gender-matched healthy volunteers served as controls. RESULTS: Both the distal and proximal ulnar a-CMAPs (P=0.001) and the MCV (P=0.014) values were significantly decreased in patients, than the controls. The sensory conduction study was normal. The ulnar F wave persistence in the ALS patients was significantly lower than that of the controls (P=0.0007). The mean (P=0.0001), minimal (P=0.0001) and maximal (P=0.0001) F wave latencies were significantly prolonged, the F wave amplitudes (P=0.0001) were significantly higher and the F wave chronodispersion (P=0.014) was significantly increased in the patients than the controls. CONCLUSIONS: Significant F wave abnormalities occur in patients with ALS, even those patients having no advanced disease. Increased F wave amplitudes combined with low persistence is a pattern consistent with ALS. SIGNIFICANCE: Our results show that patients with ALS having predominantly LMN involvement also have electrophysiological UMN dysfunction.

[Influences of decellularization processes on immunogenicity of chemically acellular nerve allografts].

OBJECTIVE: To investigate the relationship between immunogenicity and decellularization processes of chemically acellular nerve allografts. METHODS: Adult Sprague Dawley rats were used as nerve donors and adult male Wistar rats used as nerve recipient hosts. 25 mm nerve segments were excised from SD rats' sciatic nerves. The nerve segments were decellularized via an improved chemical decelluarization treatment as follows: (1) nerve segments were rinsed with cold sterile Ringer's solution; (2) stabilized by pinning the ends to a thin plastic support, and submerged in 4% Triton-100 solution 12 h; (3) soaked into 3% sodium deoxycholate for 12 h; (4) washed in distilled water for 6 h. The procedures were repeated once again. The acellular nerve allografts from SD rats were sterilized by gamma irradiation and implanted into Wistar rats subcutanously. The control group was implantation of fresh nerve allografts from SD rats. The immunogenicity of acellular nerve allograft was tested by immunohistochemical examination of the intensity of CD3(+), CD4(+) and CD8(+) cells that infiltrated the allografts. Ulnar nerve segments were obtained from forearms of dogs and decellularized according to above procedures. According as the decellularization times, The ulnar nerve segments were divided into three subgroups: in group I, group II and group III, the nerve segments were decellularized repeatedly two, three and four cycles respectively. Each ulnar nerve segment was subdivided into five portions from proximal to distal end. The degrees of decellularization, demyelination and basal lamina integrity of extracellular matrix scaffold were observed with microscope and assessed by a score system. The immunohistochemical staining of GAG was observed. RESULTS: The intensity of CD3(+), CD4(+) and CD8(+) T cells that infiltrated the allografts was greatly lower in acellular nerves than in fresh nerves. The mild cell-mediated host-graft immunorejection in acellular nerves was observed. On the decellularization procedures, the cells were completely extracted from nerves in all groups, but the myelin sheath were partially existed, and the GAG was present in the basal membrane of myelin sheath. In the score of demyelination, there were no statistical differences between groups (P > 0.05). The statistical difference of basal lamina integrity scores between group I and group II, group I and group III were significant (P < 0.05). As increasing the times of process, the degrees of disintegrity of basal lamina was significantly enhanced. CONCLUSIONS: Although decellularization processes significantly reduce the cell-mediated immunorejection of acellular nerve allografts, it can induce mild immunoreaction all the same, the antigen that responsible for immunogenicity may be the residual component of GAG in myelin sheath.

Location of motoneurones projecting to the cat distal forelimb. II. Median and ulnar motornuclei.

The position of the motornuclei projecting through the median (Mn) and ulnar (Ul) nerves to the cat distal forelimb has been investigated. Horseradish peroxidase (HRP) and fluorescent (Fl) compounds have been used as retrograde tracers. They were either injected into forelimb muscles or applied to the proximal end of transected forelimb nerves. Limb muscles that were not investigated were carefully denervated. The position and the architecture of the individual motornuclei were traced with HRP. The topographical relations between the nuclei were established with application of up to three different Fl compounds in the same animal. The Mn motoneurones had a bimodal distribution in the brachial spinal cord. The motoneurones to the pronator teres and flexor carpi radialis muscles were located in C7 and the other Mn motoneurones were located in C8 and Th1. In C7 the Mn motoneurones occupied a single representation area, which is located some distance medially of the lateral funiculus. In C8 and Th1 two Mn representation areas were found: A dorsal one that contacts the lateral funiculus and is located at the level of the central canal; a ventral one that is located ventrally in the ventral horn. The dorsal area is occupied by the motornuclei projecting to the intrinsic hand muscles and the ventral one by the nuclei projecting to the limb. The Ul motoneurones extend with an unimodal distribution from the caudal C7 to the caudal Th1 segments. They occupy a single, broad representation area. The dorsal part, which contacts the lateral funiculus, is located at the level of the central canal and harbours the nuclei to the intrinsic hand muscles. The other Ul nuclei are located ventromedially deep in the ventral horn. These results, together with those from the companion paper on the location of the deep radial motornuclei, provide important anatomical information for the investigation of the cat brachial enlargement.

Comparison of motor conduction techniques in the diagnosis of carpal tunnel syndrome.

OBJECTIVE: To compare the sensitivities of motor wrist-to-palm (W-P) conduction velocity and two median-ulnar motor latency differences with that of sensory W-P conduction velocity in the diagnosis of carpal tunnel syndrome (CTS). METHODS: This study included 116 consecutive patients with CTS (160 hands) referred for evaluation and 100 volunteers who served as controls. Median motor and sensory nerve responses with wrist and palm stimulation allowed for the determination of motor and sensory W-P CV (W-P MCV and SCV). Two motor distal latency (MDL) differences between the median-thenar and ulnar-hypothenar (M-U) muscles and between the median-second lumbrical and ulnar-interossei muscles (2L-INT) were measured and calculated. The mean values of controls plus or minus 2.5 SD served as the normal limits. RESULTS: Among the 160 hands with suspected CTS, 11 (6.88%) had normal electrodiagnostic studies and 149 (93.1%) had at least one abnormal electrodiagnostic study. Among the 149 hands with an abnormality, 139 (86.88%) had abnormal W-P MCV and 129 (80.63%) had abnormal W-P SCV. The sensitivity for 2L-INT was 77.5%, and it was 70% for M-U, 68.75% for median MDL, and 73.75% for sensory distal latency. Combining W-P MCV and W-P SCV allowed for the detection of abnormalities in 147 hands (91.88%) and yielded a markedly improved diagnostic rate compared with W-P SCV alone. CONCLUSION: Motor W-P conduction study is more valuable and no more difficult than sensory W-P conduction study for the diagnosis of CTS. In patients with suspected CTS in whom the results of conventional nerve conduction studies are normal, studying both motor and sensory W-P conduction increases the diagnostic yield.

Projection of forelimb nerve afferents to external cuneate nucleus of the rat as revealed by intraneural injection of a neurotoxic lectin, Ricinus communis agglutinin.

This study seeks to extend the observations of previous studies of projection of primary afferent fibres from the forelimb nerves and muscles to the external cuneate nucleus (ECN) of mammals using a neurotoxic lectin, Ricinus communis agglutinin (RCA) to achieve chemical ganglionectomy of the dorsal root ganglia. Following intraneural injection of RCA into the three main forelimb nerves, namely the radial, ulnar and median nerves, terminal degeneration of the primary afferent fibres in the ECN was studied under the light microscope by means of the Fink-Heimer method. The results show that the primary afferent fibres from these three nerves project to the medial part of the ECN. The field of terminal degeneration take a crescentic form. The projection from the median nerve was most dorsally located whereas that from the radial nerve was the most ventral with extensive overlaps between them. Of the three nerves, the projection from the radial nerve was the most dense. Rostrocaudally, the three nerves also show extensive overlaps. The rostrocaudal extent of maximum terminal degeneration was greatest for the radial nerve and least for the median nerve. Analysis of variance showed that these differences were statistically significant. This suggests that the radial nerve has the most extensive projection to the ECN and the median nerve the least.

Leukaemia inhibitory factor rescues motoneurones from axotomy-induced cell death.

The death of spinal motoneurones after axotomy provides a useful model for studying novel factors which prevent motoneurone loss in vivo. Peripheral nerves of newborn rats were unilaterally transected and treated with either a vehicle solution or leukaemia inhibitory factor (LIF). Compared with the vehicle controls, treatment with a gelfoam containing LIF significantly reduced motoneurone loss: from 38% to 22% after 3 days and from 55% to 38% after 7 days. The loss of motoneurones was further reduced by placing the LIF-containing gelfoam inside a silicone chamber: from 39% to 15% after 7 days, which represented a 62% rescue. Thus, LIF is a potential therapeutic agent for preventing the loss of injured or diseased motoneurones.

Cultures enriched in Schwann-like cells from dissociated nerve segments of the adult cat.

Methods for producing Schwann cell-rich cultures from nerve segments of the adult cat were investigated. Efficient dissociation was achieved with a combination of collagenase and trypsin. Differential attachment of elements within the resulting suspension allowed production of Schwann cell-rich cultures derived by outgrowth from slowly attaching segments of degenerating myelin sheath tube containing Schwann-like cells. Rapid trypsinization during subculture further increased the proportion of Schwann-like cells in these cultures to 90% or more.

Somatotopic organization of motoneurons innervating the pronators, carpal and digital flexors and forepaw muscles in the dog: a retrograde horseradish peroxidase study.

Central distribution of motor neurons innervating the individual forearm and forepaw muscle through the median and ulnar nerves was studied in the dog using the retrograde horseradish peroxidase (HRP) method. All HRP-labeled cells were seen ipsilaterally in restricted parts within the dorsolateral (DL) and retrodorsolateral nucleus (rDL) of the ventral horn at levels from the cranial tip of C7 to the cranial third of T2. The results showed that motoneurons situated more cranially supply the more proximal muscles and those situated more caudally supply the more distal ones, and that the pronator motoneurons occupy longitudinally the central part of the DL; the carpal flexor motoneurons, the medial part of the DL; the digital flexor motoneurons, the dorsal central part of the DL and the lateral fourth of the rDL; and the forepaw muscle motoneurons, the medial three-fourths of the rDL. These somatotopic arrangements of each motor pool could be correlated with the location and action of the muscles.

Double peak sensory responses at submaximal stimulation.

OBJECTIVE: The objective of the study was to obtain knowledge about the different physiological situations where a double peak sensory response normally occurs and to better understand the significance of this particular sensory response. METHODS: In 14 healthy subjects, conventional orthodromic sensory nerve conduction studies were performed on the median and ulnar nerves using submaximal stimulation. Various stimulus strengths, polarity, electrode positions and local anaesthesia were used to clarify the generation of the two peaks. RESULTS: When the cathode and the anode were independently moved in distal direction, the first and the second peaks moved distally, respectively. This occurred for conventional and reversed position of the electrode. Anodal stimulation was ineffective after local skin anaesthesia. CONCLUSIONS: Our experiments seem to indicate that the double response represents the two stimulation sites, under the cathode and the anode, respectively. Obviously the double response can only occur if different axons are stimulated under the two poles. The cathode and the anode do not seem preferably to stimulate fast or slow axons. Studies with superficial anaesthesia may indicate that the cathode stimulates the sensory nerve directly while the anode mainly stimulates superficial structures, skin sensory receptors or intradermal nerve terminals.

Different origins of low- and high-frequency components (600 Hz) of human somatosensory evoked potentials.

OBJECTIVE: Human median nerve somatosensory evoked potentials (SEPs) contain a low-amplitude (<500 nV) high-frequency (approximately 600 Hz) burst of repetitive wavelets (HFOs) which are superimposed onto the primary cortical response 'N20.' This study aimed to further clarify the cortical and subcortical structures involved in the generation of the HFOs. METHODS: 128-Channel recordings were obtained to right median nerve stimulation of 10 right-handed healthy human subjects and in 7 of them additional to right ulnar nerve. Data were evaluated by applying principal component analysis and dipole source analysis. RESULTS: Different source evaluation strategies provided converging evidence for a cortical HFO origin, with two different almost orthogonally oriented generators being active in parallel, but with a phase shift of a quarter of their oscillatory period, while the low-frequency 'N20' is adequately modeled by one tangential dipole source. Median and ulnar derived low-frequency and HFO cortical sources show a somatotopic order. Additionally, generation of the HFOs was localized in subcortical, near-thalamic and subthalamic source sites. The near-thalamic dipole was located at significantly different sites in HFO and low-frequency data. CONCLUSIONS: The cortical HFO source constellation points to a 'precortical' source in terminals of thalamocortical fibers and a second intracortical HFO origin. Furthermore, HFOs are also generated at subcortical and even subthalamic sites. Near-thalamic, the HFO and low-frequency signals are generated or modulated by different neuron populations involved in the thalamocortical outflow.

Variation in diagnostic strategy of the EMG examination--a multicentre study.

OBJECTIVES: In order to improve the universal quality of the EMG examination, knowledge about the variation among physicians is needed. METHODS: The variation among physicians in diagnostic strategy or criteria for diagnosing was analysed from a multicentre database with 940 EMG examinations sampled by seven physicians from six laboratories in Europe. RESULTS: For the whole group of patients as well as for the subgroup of patients with polyneuropathy, variation among physicians in examination techniques, number of examined structures per patient and number of abnormal structures per patient required for a diagnosis was found. Some of the variation may be explained by use of different techniques, which showed differences in sensitivity, while some of the variation may be due to differences in diagnostic strategy and criteria for diagnosing. CONCLUSIONS: The study indicates a need for development and revision of international guidelines for EMG practice although implementation of standards requires caution.

Representation of reaching and grasping in the monkey postcentral gyrus.

In area 2 and further caudal part of the alert monkey's postcentral gyrus, we found neurons which were activated preferentially or only by monkey's self-initiated hand actions to reach or grasp objects. Among them we studied those neurons which were selective to either (1) projecting the arm and stretching digits toward an object (reaching); (2) grasping with the first and the second digit (radial grasp, precision grip); (3) grasping with the ulnar digits and palmar skin (ulnar grasp, whole hand grip); or (4) scratching or touching an object with distal digit pads and nails. Neurons of the radial grasp type were found in the lateral part of the digit region while all other types of neurons were found in the medial part. Each of hand action neurons was found in a cluster of neurons which had common receptive fields corresponding to effective sites for the particular type of grasping.

Distribution of median, ulnar and radial motoneurons in the monkey spinal cord: a retrograde triple-labeling study.

Topographic distribution of motoneurons innervating hand muscles through the median (Mn), ulnar (Ul), or radial (Rd) nerves was examined using a retrograde multiple-labeling technique in the macaque monkey. The Mn and Ul motoneurons, i.e. flexor motoneurons, were distributed from C6 to T2 and from C7 to T2 segments of the spinal cord, respectively, while the Rd motoneurons, i.e. extensor motoneurons, were distributed from C4 to T2. The present study further revealed partial intermingling of the cell bodies and partial overlap of the dendritic fields among the motoneurons projecting through different nerves, indicating that subregions of motoneuronal pool participate in coordination between the flexor and extensor, or among the flexor muscles. It was suggested that there exists a control mechanism for precise hand movements in the spinal cord.

Acidic fibroblast growth factor prevents death of spinal cord motoneurons in newborn rats after nerve section.

The death of spinal axotomised motoneurons provides a useful tool for studying neurotrophic factors which could prevent motoneuron loss in vivo. Median and ulnar nerves of newborn rats were unilaterally sectioned and topically treated with either a vehicle solution or acidic fibroblast growth factor (aFGF). aFGF treatment increased the survival of the median and ulnar spinal motoneurons, after 7 days of axotomy, from 37% to 63%. These results show that aFGF is a neurotrophic factor for newborn spinal motoneurons, and suggest that this protein is a potential therapeutic agent for preventing the death of damaged motoneurons.

Regeneration through nerve allografts in the cynomolgus monkey (Macaca fascicularis).

With the use of ulnar nerves of cynomolgus monkeys, the present study examined whether basal laminae of Schwann cells can serve as conduits for regenerating axons in nerve allografts from non-human primates. A segment of ulnar nerve was transected distal to the elbow joint one week before grafting. In Group A, a distal segment of the transected nerve was transplanted, after freezing and thawing, into the ulnar nerve of another monkey, at a level that corresponded to that from which the graft was taken. In Group B (the control group), the segment of nerve was grafted in the same manner but without cryotreatment. Two weeks, five weeks, eight weeks, and five months after grafting, the graft and the host nerve were examined with light and electron microscopy. Within two weeks after grafting in Group A, after degradation of the cellular components of the Schwann cells, the basal laminae of the Schwann cells were intact in the form of tubes. Within five weeks, many regenerating axons grew out into these basal lamina tubes in the three-centimeter-long grafts and extended into the host nerve. As seen at the wrist (seven centimeters from the distal suture) five months after grafting, the axons exhibited fully mature myelination both in the graft and in the host nerve. In contrast, in Group B, in which the Schwann cells had not been disrupted by cryotreatment, cellular components and connective-tissue matrices, including basal laminae, had been degraded and had been replaced by invading cells, which filled the endoneurial spaces of the graft. Five months after grafting, axonal growth had been arrested in the graft one centimeter distal to the proximal suture. The beneficial effect in Group A appears to have been the result of the retention and preservation of intact basal laminae of Schwann cells after rapid removal of killed Schwann cells and myelin debris. Killing of Schwann cells by freezing before grafting may abolish the immune response to the Schwann cells in allografts and lead to fragmentation and disruption of myelin, which facilitates the rapid removal of myelin by macrophages.

Motor nerve topology reflects myocyte morphology in hamster retractor and epitrochlearis muscles.

Neuromuscular activation is a primary determinant of metabolic demand and oxygen transport. The m. retractor and m. epitrochlearis are model systems for studying metabolic control and oxygen transport; however, the organization of muscle fibers and motor nerves in these muscles is unknown. We tested whether the topology of motor innervation was related to the morphology of muscle fibers in m. retractor and m. epitrochlearis of male hamsters ( approximately 100 g). Respective muscles averaged 47 and 12 mm in length 100 and 35 mg in mass. Staining for acetylcholinesterase revealed neuromuscular junctions arranged in clusters throughout m. retractor and as a central band across m. epitrochlearis, suggesting differences in fiber morphology. For both muscles, complete cross-sections contained approximately 1,700 fibers. Fiber cross-sectional areas were distributed nearly normal in m. epitrochlearis (mean = 1,559 +/- 17 microm(2)) and skewed left (P < 0.05) in m. retractor (mean = 973 +/- 15 microm(2)). Single fiber length (Lf) spanned muscle length (Lm) in m. epitrochlearis, while fibers tapered to terminate within m. retractor (Lf/Lm = 0.43 +/- 0. 02). With myelin staining, a single branch of ulnar nerve projected axons across the midregion of m. epitrochlearis. For m. retractor, the spinal accessory nerve branched to give rise to proximal and distal regions of innervation, with intermingling of axons between nerve branches. Nerve bundle cross-sections stained for acetylcholinesterase indicate that each motor axon projects to an average of 65 muscle fibers in m. epitrochlearis and 100 in m. retractor. Differences in fiber morphology, innervation topology, and neuromuscular organization may contribute to the heterogeneity of metabolic demand and oxygen supply in skeletal muscle.