Evaluation of dermal myelinated nerve fibers in diabetes mellitus

Skin biopsies have primarily been used to study the non‐myelinated nerve fibers of the epidermis in a variety of neuropathies. In this study, we have expanded the skin biopsy technique to glabrous, non‐hairy skin to evaluate myelinated nerve fibers in the most highly prevalent peripheral nerve disease, diabetic polyneuropathy (DPN). Twenty patients with DPN (Type I, n = 9; Type II, n = 11) and 16 age‐matched healthy controls (age 29–73) underwent skin biopsy of the index finger, nerve conduction studies (NCS), and composite neuropathy scoring. In patients with DPN, we found a statistically significant reduction of both mechanoreceptive Meissner corpuscles (MCs) and their afferent myelinated nerve fibers (p = 0.01). This myelinated nerve fiber loss was correlated with the decreased amplitudes of sensory/motor responses in NCS. This study supports the utilization of skin biopsy to quantitatively evaluate axonal loss of myelinated nerve fibers in patients with DPN.     

Quantitative analysis of myelinated nerve fibers of peripheral nerve in streptozotocin-induced diabetes mellitus

The purpose of this study was to examine morphometric changes of myelinated fibers in early stages of experimental diabetes mellitus. Adult male Wistar rats aged 17 wk were used in this study. Diabetes mellitus was induced by streptozotocin. Samples of common peroneal nerve from diabetic rats (4 and 8 wk after induction of diabetes mellitus) and age-matched control animals were removed and processed. The semithin cross sections were stained with toluidine blue and used for myelinated fiber computer-aided morphometric analysis. There were no significant changes in diabetic animals after 4 wk duration of the disease. There was significant reduction in myelinated nerve fiber caliber in diabetic rats 8 wk after induction of diabetes as compared to age-matched controls. There was no significant reduction of axonal area in this group of diabetic rats, so diminution of fiber area was caused predominantly by reduction of myelin sheath area. The study demonstrates that the induction of diabetes mellitus in rat by streptozotocin is accompanied by early changes of the morphometric indices of myelinated nerve fibers of peripheral nerve.     

Motor fibers in the sural nerve

Summary In an ischemia-induced model of an acute motor neuron disorder, there is anterior horn cell damage with Wallerian degeneration in ventral roots; dorsal root ganglia and dorsal roots are unaffected. In a mixed nerve there is axonal degeneration reflecting loss of motor fibers. The sural nerve is normal showing that it does not contain motor fibers. This observation is relevant to the neuropathology of motor neuron disease where axonal degeneration found in the sural nerve suggests involvement of sensory fibers.Portions of this paper were presented at the 37th Annual Meeting of the American Academy of Neurology in Dallas, Texas in April 1985     

Remyelination of nerve fibers in the transected frog sciatic nerve

This project tests an important aspect of the cellular events controlling the processes of recovery of function and remyelination that follow demyelination in the peripheral nervous system. Frog sciatic nerves have been shown to survive and remain functional for up to 10 days following transection. We have utilized this property in order to dissociate the recovery process from possible control by the neuronal soma. Xenopus sciatic nerves were demyelinated in one branch by an intraneural injection of lysolecithin. The nerve was cut proximally to the injection site either immediately before, or several days after the lysolecithin injection. Recovery of function and remyelination were then followed by electrophysiological, optical, and ultrastructural techniques applied both to whole branches and single fibers. Controls included the cut but uninjected branch, and injected but uncut nerves. The progression of events during both demyelination and recovery in cut axons was indistinguishable from that in uncut fibers. This suggests that this process may be under local control and can be initiated and carried out in the absence of constant communication with the nerve cell body.     

Distribution of OL-protocadherin in axon fibers in the developing chick nervous system

OL-protocadherin (OL-pc) is a homophilic cell adhesion molecule that belongs to the cadherin gene superfamily. We cloned and characterized the chicken homologue of OL-pc and examined its expression pattern in chick embryos mainly from embryonic day (E) 3.5 to E6.5. The structure of chick OL-pc was found to be essentially the same as that of mammalian OL-pc's except for some small deletions and insertions in the amino acid sequence. OL-pc protein was detected prominently along developing axonal fibers in the brain and also in the peripheral nervous system. In addition, it was detected in some mesenchymal cells and in the embryonic ectoderm of the mandible and limb bud. In the spinal cord, OL-pc was specifically expressed in motor neurons, and the protein was distributed along motor nerves. Motor nerves merged gradually with sensory nerves showing negative/faint OL-pc expression, but their fibers remained separated as small bundles in the nerves. Interestingly, OL-pc-positive motor nerves such as those to the sternocoracoideus became segregated from OL-pc-faint/weak motor nerves at the plexus region. Moreover, OL-pc was distributed along the path of the branchial nerves. These results suggest that OL-pc might play some roles in axon navigation such as in axon elongation, selective fasciculation, and pathfinding in the early stage of neural development.     

Expression of growth-associated protein 43 in the skin nerve fibers of patients with type 2 diabetes mellitus

The growth-associated protein 43 (GAP-43) is known as a marker of regenerating nerve fibers and their continuous remodeling in the adult human skin. The purpose of this pilot study was to investigate a possible role for GAP-43 in the detection of the early stages of small-fiber neuropathy in patients with type 2 diabetes mellitus (DM2) as compared with a well- established and validated parameter - intra-epidermal nerve fiber density (IENFD) of protein gene product 9.5 (PGP 9.5) immunoreactive intra-epidermal C fibers. In a group of 21 patients with DM2 within three years of diagnosis (13 men, 8 women; mean age 53.9±12.8; range 30-74) and a group of 17 healthy volunteers (8 men, 9 women; mean age 55.8±8.5; range 45-70 years), skin punch biopsies were taken from a distal calf and double immunostained with both PGP 9.5 and GAP-43. In healthy controls, 96.8% of 629 PGP 9.5 immunoreactive fibers were immunostained with GAP-43; the proportion of PGP 9.5 intra-epidermal nerve fibers immunoreactive for GAP-43 in control subjects ranged from 86.5 to 100%. In DM2 patients, IENFD was significantly lower compared to controls (median, 1.5 vs. 11.2/mm; p<0.001). The proportion of GAP-43 immunoreactive intraepidermal nerve fibers was significantly lower in DM2 patients compared to healthy controls (73.6% of 337 PGP 9.5 positive fibers; p<0.001); ranged from 0 to 98.1%. In conclusion, these results show that impaired regeneration of intra-epidermal C fibers in the early stages of type 2 diabetes mellitus, as indicated by GAP-43, might be a marker of incipient diabetic neuropathy.     

Validation of the nerve axon reflex for the assessment of small nerve fibre dysfunction

Objective

To validate nerve–axon reflex‐related vasodilatation as an objective method to evaluate C‐nociceptive fibre function by comparing it with the standard diagnostic criteria.

Methods

Neuropathy was evaluated in 41 patients with diabetes (26 men and 15 women) without peripheral vascular disease by assessing the Neuropathy Symptom Score, the Neuropathy Disability Score (NDS), the vibration perception threshold (VPT), the heat detection threshold (HDT), nerve conduction parameters and standard cardiovascular tests. The neurovascular response to 1% acetylcholine (Ach) iontophoresis was measured at the forearm and at both feet by laser flowmetry. An age‐matched and sex‐matched control group of 10 healthy people was also included.

Results

Significant correlations were observed between the neurovascular response at the foot and HDT (r_s = −0.658; p<0.0001), NDS (r_s = −0.665; p<0.0001), VPT (r_s = −0.548; p = 0.0005), tibial nerve conduction velocity (r_s = 0.631; p = 0.0002), sural nerve amplitude (r_s = 0.581; p = 0.0002) and autonomic function tests. According to the NDS, in patients with diabetes who had mild, moderate or severe neuropathy, a significantly lower neurovascular response was seen at the foot than in patients without neuropathy and controls. A neurovascular response <50% was found to be highly sensitive (90%), with a good specificity (74%), in identifying patients with diabetic neuropathy.

Conclusion

Small‐fibre dysfunction can be diagnosed reliably with neurovascular response assessment. This response is already reduced in the early stages of peripheral neuropathy, supporting the hypothesis that small‐fibre impairment is an early event in the natural history of diabetic neuropathy.Diabetic neuropathy includes nerve fibres with both small and large diameter.¹ Small‐fibre neuropathy remains a diagnostic challenge because currently available techniques are not objective, have a high variability and are not routinely applied.^2,3 Consequently, the diagnosis of small‐fibre neuropathy can easily be missed.Assessment of nerve–axon reflex‐related vasodilatation, or neurovascular response, has been proposed as an objective method to quantify C‐nociceptive fibre function.⁴ Activation of peripheral C‐fibres by different noxious stimuli, or activation experimentally by acetylcholine (Ach), leads to the conduction of the impulse, both orthodromically to the spinal cord and antidromically to other branches of the same C‐fibres, which then release vasodilating neuropeptides. This vasodilative response is part of Lewis''s triple anti‐inflammatory response.⁵This neurovascular response is impaired in patients with diabetic neuropathy.⁶ Moreover, local anaesthesia markedly reduces the neurovascular response in controls and patients with diabetes without peripheral neuropathy, whereas in patients with diabetic peripheral neuropathy, the already low neurovascular response at the foot does not decrease further after the induction of local anaesthesia.⁷As all previous findings indicate that C‐fibre function is the main determinant of the neurovascular response, it is reasonable to hypothesise that the assessment of this response may be used as a surrogate measure of C‐fibre integrity. The aim of the present study was therefore to validate the neurovascular response for the assessment of small‐fibre function by comparing it with the currently used techniques.     

Demonstration of axon reflexes in human motor nerve fibres

Responses of single muscle fibres to electrical stimulation of the tibial nerve trunk or of the intramuscular nerve twigs were detected in young volunteers without evidence of neurological disease. With suitably adjusted amplitude of the stimulus, clear-cut double distribution of the response latencies was obtained in some fibres. Experiments with two stimulating cathodes and with recordings from more than one muscle fibre in the same motor unit suggest that axon reflexes were involved. The results indicate that axonal branching normally occurs not only in the intramuscular course of the nerve but also outside the muscle, in some cases even rather high in the nerve trunk. The possibility is discussed that axon reflexes may underlie fasciculations evoked by neostigmine and those seen in some other conditions, such as amyotrophic lateral sclerosis, as well as muscle cramps in normal subjects.     

Effect of acclimation temperature on the axon and fiber diameter spectra and thickness of myelin of fibers of the optic nerve of goldfish

The optic nerves of common goldfish acclimated to 5 and 25 degrees C were fixed with glutaraldehyde in either phosphate buffer or PIPES with EGTA, post-fixed with osmium tetroxide, and examined by electron microscopy. The axon diameter spectra, from axons measured in electron micrographs and those measured on the electron microscope screen, differ noticeably with acclimation temperature. At the lower temperature, there is a definite shift toward the occurrence of larger fibers compared with the spectrum of the 25 degrees C fish. Although the number of fibers assessed is small compared with the total number in the goldfish nerve, these results confirm our previous study. These findings could be attributed to an increase in the number of new fibers during the acclimation to the higher temperature. We discuss this possibility and on the available evidence find it unlikely. Other changes in the axon and fiber are also seen with acclimation temperature. The axon to fiber diameter ratio, made directly from the electron micrographs, shows that axons from the nerves of the higher acclimation temperature fish possess consistently thicker myelin sheaths than are found for axons in nerves of the lower temperature fish. This finding is also in agreement with results obtained by us from measurements independent of each other.     

Effects of age on nerve fibers in the rhesus monkey optic nerve

During normal aging there is a reduction in white matter volume in the cerebral hemispheres and structural abnormalities in myelin in some parts of the central nervous system, but whether nerve fibers are lost with age and whether the myelin changes are ubiquitous is not known. Studying the optic nerve, which is a circumscribed bundle of nerve fibers, offers an opportunity to gain further insight into the effects of normal aging on white matter. The present study examined the optic nerves from young (4-10 years) and old (27-33 years) rhesus monkeys using light and electron microscopy. These nerves had been perfused transcardially to obtain optimal preservation of the tissue. Varying degrees of degeneration were encountered in all the optic nerves from the old monkeys. The changes included myelin abnormalities, similar to those reported in other parts of the central nervous system; the presence of degenerating axons and their sheaths; changes in neuroglial cells; and thickening of the trabeculae of connective tissue in the nerve. The total number of nerve fibers was reduced from an average of 1.6 x 10(6) in the young optic nerves to as few as 4 x 10(5) in one old monkey, and with one exception in all of the old optic nerves the packing density of nerve fibers was less than in any of the young optic nerves. The degenerative changes were most marked in those optic nerves that contained the fewest nerve fibers.     

Intrinsic nerve fibers of the primate endometrium

An experiment was designed to test the currently favored theory that inasmuch as nerve fibers are undemonstrable in the outer glandular portion of the uterine mucosa certain endometrial functions must be regulated solely by humoral mechanisms. Searches ordinarily have been confined to tissues removed surgically which have begun to undergo post-mortem changes. A methylene-blue intravital staining method, quite specific for fine peripheral nerve fibers, was used in five anesthetized cats, a guinea pig, and three monkeys; injections of the dye solution were made directly into the superficial glandular layer of the living endometrium. Similar injections were made in pieces of endometrium that had been removed surgically 5 to 10 min previously. Fine nerve fibers were found in all instances, but they stained more sharply and deeply after intravital injections. The presence of neurons in a region usually considered to be exclusively under humoral control may require modifications of current theories. In species that menstruate, the endometrial nerve fibers probably undergo cyclical degeneration and regeneration physiologically.     

Iron deficiency disrupts axon maturation of the developing auditory nerve

Iron is critical in multiple aspects of CNS development, but its role in neurodevelopment--the ability of iron deficiency to alter normal development--is difficult to dissociate from the effects of anemia. We developed a novel dietary restriction model in the rat that allows us to study the effects of iron deficiency in the absence of severe anemia. Using a combination of auditory brainstem response analyses (ABR) and electron microscopy, we identified an unexpected impact of nonanemic iron deficiency on axonal diameter and neurofilament regulation in the auditory nerve. These changes are associated with altered ABR latency during development. In contrast to models of severe iron deficiency with anemia, we did not find consistent or prolonged defects in myelination. Our data demonstrate that iron deficiency in the absence of anemia disrupts normal development of the auditory nerve and results in altered conduction velocity.     

Beading of myelinated nerve fibers

The phenomena of beading upon stretching was studied in nerve fibers of freeze-substituted nerves. Beading appears rapidly along the length of the mammalian nerve. Nerve fibers of frogs kept at low temperatures have a crenated shape in cross section and show wrinkling in longitudinal section, but no beading is produced upon stretching them. Frogs kept at higher temperatures have nerve fibers which are more cylindrical in shape and a moderate degree of beading can then be excited by stretching. The beading is due to constrictions along the length of the fibers. Electron micrographs show an increased concentration of neurofilaments in the constricted part of the fiber, a change interpreted as squeezing of the fluid part of the axoplasm into the expanded parts of the beads.     

Histamine-immunoreactive nerve fibers in the mammalian spinal cord

New sensitive antisera against histamine were used to study the distribution of histamine-immunoreactive nerve fibers in the spinal cord of several mammalian species. Tissues were fixed with carbodiimide by transcardiac perfusion or immersion. A few immunoreactive nerve fibers were found in the cervical spinal cord of the rat in the superficial laminae of the dorsal horn, around the central canal and scattered in the anterior horn. The density of immunoreactive fibers in the cervical spinal cord of the guinea pig and tree shrew was higher, but still low. The densest networks of histamine-immunoreactive fibers were seen in the cervical spinal cord of the pig. The laminar distribution of histamine-immunoreactive fibers was similar in all species. Histamine-immunoreactive fibers were densest in lamina X, followed by laminae I-II. Scattered fibers were also seen in the white matter in the lateral and posterior funiculus in the pig. In the rat and the guinea pig, no histamine-immunoreactive cell bodies were seen in the spinal sensory ganglia. The results suggest that the histamine-immunoreactive nerve fibers in the spinal cord may originate from the brain, probably from the posterior hypothalamus, and the fiber projection is more extensive in higher mammalian species. The role of histamine in the spinal cord is not known, but it may be involved in, e.g., pain sensation.