We have established a reference range for intraepidermal nerve fiber density in normal humans by means of a simple quantitation method based on enumeration of individual intraepidermal nerve fibers on vertical sections of punch skin biopsy specimens stained with the sensitive panaxonal marker anti-protein gene product 9.5. The utility of the density measurement was confirmed for sensory neuropathy with a diagnostic efficiency of 88%. Skin biopsies may be useful to assess the spatial distribution of involvement in peripheral nerve disease and the response to neurotrophic and other restorative therapies.

This study examined Schwann cell behavior during paranodal demyelination induced by beta,beta'-iminodipropionitrile (IDPN). The stimuli for Schwann cell proliferation, extensively studied in vitro, are less well understood in vivo. Most in vivo systems previously used to examine Schwann cell proliferation in disease are dominated by loss of internodal myelin sheaths. As used in this study, IDPN administration produces neurofilamentous axonal swellings and paranodal demyelination, without segmental demyelination or fiber degeneration. We asked whether Schwann cells would proliferate following the restricted paranodal demyelination that accompanies the axonal swellings, and if so what the sources and distributions of new Schwann cells might be. IDPN was given as a single large dose (2 ml/kg) to 21-d-old rats. Neurofilamentous axonal swellings formed in the proximal regions of motor axons, reaching their greatest enlargement in the root exit zone 8 d after IDPN administration. These swellings subsequently migrated distally down the nerves at rates approaching 1 mm/d. The axonal enlargement was consistently associated with displacement of the myelin sheath attachment sites into internodal regions, and consequent paranodal demyelination. This stage was associated with perikaryal changes, including nucleolar enlargement, "girdling" of the perikaryon, and formation of attenuated stalks separating the perinuclear region from the external cytoplasmic collar. Schwann cells proliferated abundantly during this stage. Daughter Schwann cells migrated within the endoneurial space (outside the nerve fiber basal laminae) to overlie the demyelinated paranodes of swollen nerve fibers. In these regions, local proliferation of Schwann cells continued, resulting in large paranodal clusters of Schwann cells. As the axonal calibers subsequently returned to normal, the outermost myelin lamellae of the original internodes returned to their paranodal attachment sites and the supernumerary Schwann cells disappeared. Formation of short internodes, segmental demyelination, and nerve fiber loss were rare phenomena. These results indicate that paranodal demyelination is a sufficient stimulus to excite abundant Schwann cell proliferation; neither internodal demyelination nor myelin breakdown is a necessary stimulus for mitosis. The 3H-thymidine incorporation studies indicated that the sources of new Schwann cells included markedly increased division of the Schwann cells of unmyelinated fibers and, as they formed, supernumerary Schwann cells. In addition, there were rare examples of 3H-thymidine incorporation by Schwann cells associated with myelinated nerve fibers.(ABSTRACT TRUNCATED AT 400 WORDS)

It is of interest to quantify accurately lineal biological features such as nerve fibres, capillaries, and tubules for studies of development and diseases such as sensory neuropathy, and for evaluation of therapeutic regimens in humans and animal models. An unbiased stereological method to sample and estimate total length of immunostained epidermal nerve fibres by using vertical sections of punch skin biopsies from two human volunteers is presented. The essential steps in the procedure are as follows: (1) serially section the skin punch in a random plane perpendicular to the cutaneous surface; (2) immunostain a known fraction of total sections with antibody specific for nerve fibres; (3) orient a test line-grid over the epidermis; and (4) count intersections between test lines and immunostained epidermal nerve fibres. The optical fractionator method is employed to estimate total length of immunostained epidermal nerve fibres in the biopsy. By using these techniques the total length of nerve fibre in a defined region can be determined without methodological bias, assumptions or correction factors.

Schwann cell division, meticulously regulated throughout development, occurs at an extremely low level in normal adult nerves. Loss of the myelin sheath in disease results in active proliferation of Schwann cells. The dividing cells are usually thought to be the Schwann cells of the demylinated fibres and their daughters. In this study we asked if other populations of Schwann cells might also divide following focal monophasic demyelination, and if the proliferating Schwann cells would be found only in the foci of demyelination. [3H]thymidine incorporation was examined by autoradiography at intervals after topical application of lysolecithin (lysophosphatidyl choline) to rat sciatic nerves. The postlabelling intervals were set to identify premitotic cells, cells shortly after mitosis (perimitotic cells) and postmitotic cells, as well as to provide cumulative labelling over 3 days. The affected nerves had three distinct zones. The first was a zone of nearly complete demyelination immediately beneath the perineurium. The subjacent zone was normal morphologically except for numerous supernumerary Schwann cells, displacement of some Schwann cell perikarya, ultrastructural changes in a few myelinated fibres, and rare demyelinated and remyelinated fibres. The third zone, beneath the first two, was normal. In the focus of demyelination there were large numbers of Schwann cells in S phase on days 4 and 6. These cells included premyelinating Schwann cells that were contacting or ensheathing demyelinated axons or collateral axonal sprouts. The subjacent region also contained dividing Schwann cells, most of which were Schwann cells of unmyelinated Remak fibres. In addition, occasional Schwann cells of thickly myelinated fibres (fibres that had not previously undergone demyelination) were labelled by the premitotic schedule; most of these fibres had morphological abnormalities in the Schwann cell perikaryon or myelin sheath. In many, the perikaryon of the Schwann cell was beginning to separate from the rest of the Schwann cell cytoplasm and the myelin sheath. These changes suggested that these fibres were destined to undergo subsequent demyelination, a hypothesis supported by the absence of any normal myelinated fibres with labelled Schwann cell nuclei in nerves removed 1 week after labelling. Thus, this model provided no evidence for division by Schwann cells that continued to maintain myelin sheaths. Taken together, these results suggest that there is a 'surround' of Schwann cell proliferation around foci of demyelination; in this surround multiple populations of Schwann cells are recruited to proliferate, including Schwann cells of intact unmyelinated fibres. Structurally normal unmyelinated fibres appear to provide an unexpected source of new Schwann cells in nerve disease.