Alfred C. Lais

Peripheral axotomy induces neurofilament decrease, atrophy, demyelination and degeneration of root and fasciculus gracilis fibers

We have recently shown that peripheral axotomy by hindlimb amputation in adult cats sequentially results in neurofilament and microtubule decrease and axonal atrophy, myelin wrinkling, myelin remodeling (de- and remyelination), more atrophy and axonal degeneration in proximal sciatic and L7 segmental nerve fibers. The neuropathologic, morphometric and teased fiber alterations in the myelinated fibers (MF) of roots and sampled levels of fasciculus gracilis in groups of adult cats 24 months after hindlimb amputation have now been studied. We found: a severe decrease of neurofilaments, axonal atrophy, myelin wrinkling, de- and remyelination and axonal loss in posterior root axons; that these morphologic abnormalities extended up the fasciculus gracilis in the appropriate territories established from degenerative studies; that the retrograde effect was less severe in ventral root fibers, although atrophy and sprouting were demonstrated here, and that the cellular sequence of retrograde atrophic degeneration of ascending axons was similar to that observed in proximal stump axons. These findings confirm that primary afferent neurons are more vulnerable to axotomy than lower motor neurons and may provide an additional explanation for the poorer functional restoration of sensory than of motor deficit after root compression and in delayed nerve reconnection. Our observations also have important implications for interpretation of neuropathologic alterations in roots and fasciculus gracilis, since the observed features may be secondary to axotomy of peripheral nerve fibers induced by disease and not evidence of a primary derangement.

Axonal caliber and neurofilaments are proportionately decreased in galactose neuropathy.

Feeding galactose to rats induces nerve conduction abnormalities, increased levels of nerve galactitol, endoneurial edema, elevated pressure and hypoxia of endoneurial fluid, and pathological abnormalities of nerve fibers. To investigate the cellular mechanisms of the fiber lesions and their possible relationship to alterations in the nerve microenvironment, rat peroneal nerves were morphometrically evaluated eight months after the commencement of galactose feeding. Whereas the density of neurofilaments (NF/μm2) in the transverse axonal area of myelinated fibers was not significantly different between the nerves of galactose-fed and control rats, axonal areas and the number of NF/axon, when related to myelin spiral length, were significantly less in nerves of galactose-fed rats. Myelin alterations, characteristic of axonal atrophy, were also significantly increased. The present data provide evidence of a proportionate decrease in axonal caliber and the number of NF/axon in myelinated fibers in experimental galactose neuropathy, suggesting that galactose induces either decreased NF synthesis, assembly or transport. The possible role of microenvironmental alterations, including endoneurial hypoxia and hyperosmolarity, in the production of this axonal atrophy is discussed.

Number, size, and class of peripheral nerve fibers regenerating after crush, multiple crush, and graft

Morphometric characterization of fiber regeneration in a distal nerve after focal proximal nerve injury may provide useful clinical information and insights about underlying neurobiologic mechanisms. The myelinated (MF) and unmyelinated (UF) fibers of peroneal nerve of groups of mice were assessed 9 months after crush, graft, and multiple crush injury of the proximal sciatic nerve: number and size distribution of axon areas, myelin areas, and fiber diameters. After crush, number of regenerated MF and UF was almost identical to that of controls. Their size distribution had almost returned to normal. After graft and multiple crush, fiber number had returned to normal or was significantly increased beyond normal but there were only a few large fibers present. This may be explained by: (a) disproportionate regeneration of small-diameter compared to large-diameter classes of fibers; (b) misdirected regrowth of fibers, so that functional reinnervation was not established, resulting in failure of development or retrograde atrophy and degeneration; or (c) cellular alterations at the site of injury or in the distal nerve which inhibited neural outgrowth or elongation or did not inhibit outgrowth but retarded or prevented maturation. We conclude that explanation (b) is involved, and that there is some evidence favoring the roles of (a) and (c).

Technique assessment of demyelination from endoneurial injection

Abstract Solutions or sera can be subperineurally injected into rat nerve to test their neuropathologic and, especially, their demyelinating effects. In the present study we assessed the effect of the endoneurial injection technique itself. The rate of axonal degeneration and segmental demyelination increased wth extraneous movement (of the needle or the animal), increasing needle size, rapid injection, and large volumes of injectate. We showed that graded evaluation of the pathologic abnormalities of teased fibers provides a more sensitive measure of demyelination than does evaluation of transverse sections. Transverse sections provide an inadequate approach to assess the rate of axonal degeneration. Using these approaches, no difficulty was encountered in demonstrating the greatly increased demyelinating activity of 5 nmol lysolecithin (lysophosphatidylcholine) compared with lactated Ringers solution.

Ultrastructural alterations of primary afferent axons in the nucleus gracilis after peripheral nerve axotomy

Alterations of primary afferent axons in the nucleus gracilis were studied at three weeks and four, 12, and 24 months after peripheral nerve axotomy by hindlimb amputation at the hip joint in 19 female cats. The contralateral side and two cats not subjected to amputation were controls. We observed two major types of fiber alterations. One, fibers showed changes of axonal atrophy, myelin remodelling, and degeneration. Adaxonal invagination occurred more frequently (p<0.005) at three weeks postamputation, in territories known to contain centrally directed axons of primary afferent neurons of the lower limb, than in controls. Perhaps adaxonal sequestration contributed to axonal attenuation. Two, there were filamentous, granular, central core, and other types of axonal swellings found in territories containing central axons of primary afferent neuron terminals of the lower limb. These changes occurred most frequently at 12 and 24 months and were significantly more frequent than in control tissue. These reactive/dystrophic axons therefore were associated with permanent axotomy, but we have not established that they occur in central axons of primary afferent neurons, or whether they are degenerative or abortive regenerative changes.

Lipid organization in peripheral nerve: Natural abundance carbon-13 NMR spectroscopy on intact tissues

Summary Native structures of peripheral nerve myelin can directly be observed by carbon-13 nuclear magnetic resonance (NMR) spectroscopy after dissecting epineurial and perineurial tissue sheaths from the endoneurium. NMR spectra of the intact endoneurial tissue describe a highly ordered and largely immobilized lipid phase in the myelin membrane which is in marked contrast to the fluid-like lipid distribution in the intracellular lipid droplets of epineurium and perineurium. Membrane reconstitution experiments with total endoneurial lipids, but without protein, suggest a primary role of the lipids in myelin bilayer assembly. Reconstitution experiments also indicate that polar lipid-protein interactions in native myelin involve the CH2-N function of choline phospholipids, in particular.

Nerve xenografts to apportion the role of axon and Schwann cell in myelinated fiber absence in hereditary sensory neuropathy, type II

Sural nerve fascicles from two patients with a recessively inherited sensory neuropathy—hereditary sensory neuropathy type II (HSN II) with complete absence of myelinated fibers—were grafted into the left sciatic nerves of 30 nude mice. Nerve fascicles from age-matched healthy control subjects were grafted into the right sciatic nerves of the same mice. At 6 months, abundant myelinated fibers were found in the 10 disease xenografts studied—by visual microscopic examination not different from the paired control xenografts. The number of myelinated fibers, the size distribution of diameters of myelinated fibers, the slope and intercept of linear regression of number of myelin lamellae on area of axis cylinders of myelinated fibers, number of endoneurial nuclei (mostly Schwann cells) per cubic millimeter, and labeling indices of Schwann cell nuclei (with the use of [3H]-thymidine) did not significantly differ in disease and control xenografts. These studies indicate that the Schwann cells of HSN II can myelinate mouse axons. The absence of myelinated fibers in sensory nerves of HSN II is therefore due to an axonal abnormality—possibly a failure of development or degeneration in utero. The studies further indicate that grafted Schwann nerves of patients with cells from only unmyelinated fibers are the likely source of cells that ensheath and myelinate regenerating mouse axons. This provides further suggestive evidence for the role of axons in determining whether Schwann cells become differentiated into cells forming myelin of myelinated fibers or cells forming sheath cells of unmyelinated fibers.