Cellular mechanisms of rejection and regeneration in peripheral nerve allografts.

A model of rejection and regeneration of peripheral nerve allografts in rats is presented. A 2.5-cm segment of 28 right sciatic nerves was transplanted orthotopically from LEW.1W to DA and from DA to LEW.1W. With a microsurgical technique, proximal and distal coaptations were performed. In an autologous control group the same surgical procedure was applied. Evaluation included clinical estimation of motor recovery and macroscopic appearance of the graft, electrophysiological examination, conventional histology, and immunohistology. The latter concentrated on demonstration of monomorphic and polymorphic determinants of MHC class I and II antigens and of macrophages. By functional, electrophysiological, and histological parameters it was demonstrated that after rejection a certain degree of regeneration took place in the allografts. Both rejection and subsequent regeneration were studied in detail by immunohistology. During the course of Wallerian degeneration MHC class I expression on myelin sheaths could be demonstrated. When the rejection response occurred, additional MHC class II expression on myelin sheaths and on vascular endothelial was observed. Recipient specific class I-positive macrophages were infiltrating the graft from the epineurium and the coaptation sites, and were later present at the sites of myelin degradation. At 6 weeks postoperatively donor-specific MHC products were no longer detectable, but recipient-specific Schwann cells were present in the allograft tissue. We conclude that a rejection response renders a peripheral nerve allograft acellular but does not destroy the nerve architecture, still enabling it to function as an axon conduit. The regeneration in the rejected allograft however lacks the positive neurotropic and -trophic influence physiologically provided by viable Schwann cells.

Effects of limited postnatal ethanol exposure on the development of myelin and nerve fibers in rat optic nerve.

This study was designed to morphologically evaluate the effects of limited postnatal alcohol exposure on the development of myelin and axons in the rat optic nerve. Rat pups were artificially reared on Days 5-18 with a supplemented milk diet fed via a chronic gastrostomy tube. Experimental animals received 4% ethanol in their diet on Days 5-9, otherwise the experimental and control animals received identical diets in identical volumes. Optic nerve tissues were prepared for electron microscopy on Days 10, 16, 22, 29, and 90. The cross-sectional areas of optic nerves were smaller, there were fewer myelinated nerve fibers per unit area, and the progress of myelination was slowed on Day 10 in the ethanol-exposed animals. All of these effects were compensated for at later times. The ratio of myelin thickness to axon diameter was similar in experimental and control animals, indicating that the interaction between axon size and myelin formation was not affected by alcohol. The general distribution of axon sizes was unaffected by ethanol except at 10 days when the largest fibers were smaller. There was no evidence of alcohol-induced degeneration of axons, myelin, or glial structures. Thus, alcohol exposure during myelin development causes a delay in myelin acquisition that is later compensated for.

MR evaluation of early myelination patterns in normal and developmentally delayed infants.

This study demonstrates the ability of MR imaging to show progression of myelination in 64 infants and young children (ages 4 days to 36 months). T2-weighted spin-echo pulse sequences, frequently used for routine screening of intracranial disease, were used. Gray-white matter differentiation was seen in all patients, and changes occurring with age were documented. Three distinct patterns were seen, and age ranges were established for each pattern in developmentally normal children: (1) infantile (birth-6 months); (2) isointense (8-12 months); and (3) early adult (10 months onward). There was a statistically significant difference between the age ranges of the normal and developmentally delayed children showing all three patterns. These data should be helpful for identifying and following sequentially both infants with clinically suspected developmental delay and those with dysmyelinating or demyelinating disease.

Adjustment of the myelin sheath to axonal atrophy in the rat spinal root by the formation of infolded myelin loops.

Atrophy of the L4 dorsal and ventral spinal roots was experimentally induced by unilateral sciatic neurectomy in groups of young (2 and 4 months) and older (12 months) albino rats. During the 4 months following neurectomy, the occurrence of infolded myelin loops (IMLs) was quantitatively examined in transverse sections prepared using perfusion fixation with glutaraldehyde and embedding in epoxy resin. The number of IMLs was higher on the operated side and increased with the time of survival and the age of the animals. The formation of IMLs is a characteristic early response of a large-caliber myelin sheath to axonal atrophy, probably reflecting the presence of redundant myelin.

The fiber caliber of 5-HT immunoreactive axons in the dorsolateral funiculus of the spinal cord of the rat and cat.

Although there is considerable evidence that the analgesic action of electrical brain stimulation is mediated in part by serotonergic (5-HT) axons in the dorsolateral funiculus (DLF) of the spinal cord, studies in the rat have questioned the existence of this pathway. In this study, we used antisera directed against a conjugate of 5-HT and bovine serum albumin (BSA) to identify immunoreactive 5-HT axons in the DLF of the rat and cat. Both light and electron-microscopic studies were performed so that the fiber caliber of the labeled axons could also be determined. We found a rich complement of immunoreactive 5-HT axons in the DLF of both rat and cat. Although these could be seen without difficulty in the normal cat, in the rat it was necessary to make a lesion of the DLF to build up the staining rostrally. Ultrastructural analysis established that almost all of the labeled axons (in rat and cat) were unmyelinated. We conclude that there are indeed 5-HT immunoreactive axons in the DLF of the rat and cat. These presumably derive from neurons of the medullary nucleus raphe magnus (NRM), which have been implicated in the descending controls exerted by opiates and electrical brain stimulation. The results suggest that previous physiological studies of the properties of the opiate-responsive, spinally projecting NRM neurons were not made from those that are 5-HT containing.

Sensory, motor and sympathetic neurons forming the common peroneal and tibial nerves in the macaque monkey (Macaca fascicularis).

The motoneurons, dorsal root ganglion (DRG) and sympathetic ganglion (SG) cells forming the common peroneal (CPN) and tibial (TN) nerves of young and semiadult monkeys (Macaca fascicularis) were localised by the horseradish peroxidase method of tracing neuronal connections. The motoneurons forming the CPN occur in the L4-L6 segments, appearing as 1-3 groups and occupying the retroposterolateral (rpl), posterolateral (pl) and central (c) groups of motor nuclei. The motoneurons forming the TN occur in the L4-L7 segments, appearing as 1-4 groups and occupying the rpl, pl, c and anterolateral (al) groups. The motoneurons and DRG cells forming the CPN show peak frequencies at the L5 level, and the SG cells forming the same nerve, at the L6 level in most cases. The motoneurons and DRG cells forming the TN show peak frequencies at the L6 level and the SG cells forming the same nerve, also at the L6 level in most cases. The bulk of motoneurons, DRG and SG cells forming the CPN and TN are concentrated in two segmental levels. For CPN the motoneurons measure between 14-76 micron in their average somal diameters and for TN, 16-70 micron. The majority of them (65.5% for CPN motoneurons and 72% for TN motoneurons) have average somal diameters greater than 38 micron. The size spectrum of the DRG cells forming the CPN is similar to that of DRG cells forming the TN, being 12-78 micron for CPN and 10-76 micron for TN. The sympathetic neurons forming the CPN (measuring 10-44 micron) have a larger size spectrum than those forming the TN (measuring 6-33 micron). The diameter spectrum (3-20 micron for TN and 2-19 micron for CPN) and peak frequency distributions (10 micron for both TN and CPN) of the myelinated fibres present in the CPN and TN are also similar, with the CPN fibres skewing towards a slightly larger size. Many of the fibres in the young and semi-adult monkeys are not yet myelinated.

Morphometric studies of sural nerve in childhood.

Sural nerve myelinated fiber density and myelinated fiber diameter distribution have been examined in 27 control subjects, ranging in age from 1 day to 59 years. Total transverse fascicular area was measured in 10 of the subjects. There is an exponential decline in myelinated fiber density from birth to adult life. The predicted normal density (D) at any age may be derived from the formula D = (1 X 10(3]/(0.0699 + 0.00725 square root t). The distribution of myelinated fiber diameters is unimodal in the first 4 months of life, and there is a definite bimodal distribution by 2 years of age. Total transverse fascicular area of sural nerve increases progressively from values of about 0.25 mm2 in the first week to about 0.82 mm2 at 9 years. Control values for sural nerve morphometry in childhood are essential for accurate interpretation of biopsies in patients with peripheral neuropathy.

Developmental features of the neonatal brain: MR imaging. Part I. Gray-white matter differentiation and myelination.

To establish the normal appearance of the neonatal brain, 51 neonates, 29-42 weeks postconception, underwent magnetic resonance (MR) imaging with a 0.6-T magnet in a prospective study. T1-weighted images were used to devise stages for the appearance of gray-white matter differentiation and extent of myelination. The results show that from 29 to 42 weeks postconception, changes in gray-white matter differentiation and myelination follow the stages in an orderly and predictable fashion. Changes in white matter intensity appear related to progressive decrease in brain water content. Myelination progresses cephalad from the brain stem at 29 weeks to reach the centrum semiovale by 42 weeks. Delayed myelination, defined as the absence of myelin in the corona radiata by 37 weeks, was seen in nine infants with complicated perinatal courses. Awareness of these developmental features should help to minimize misinterpretation of normal changes in the neonatal brain and lead to earlier detection of pathologic conditions, both with MR imaging and computed tomography.

Characteristic variations of relative myelin sheath thickness in 11 nerves of the rat.

Computer-assisted measurements of relative myelin sheath thickness (the g ratio) were made in 11 peripheral nerves of the rat. The scatter diagrams showed nerve-specific variations in the distribution of relative myelin sheath thickness. Myelinated fibers of less than 3.5 microns axon diameter had relatively thin myelin sheaths, particularly in the splanchnic, vagus and glossopharyngeal nerves. The oculomotor nerve had two fiber populations clearly set apart in terms of relative myelin sheath thickness. Thickly myelinated fibers were found in facial and hypoglossal nerves. No single functional modality was evident for the thinly myelinated fibers.

A rapid silver-free method for staining the myenteric plexus.

A method is presented for the relatively rapid demonstration of the myenteric plexus. Saturated Sudan black B in 70% ethanol followed by 0.01% aqueous buffered thionein were used on intestinal peels (whole-mounts) to stain myelinated and unmyelinated fibers and neuron cell bodies, respectively. In contrast to accepted silver methods, these two kinds of fibers were distinguished clearly; Schwann cell nuclei and nodes of Ranvier were visible. Preparations had the following attributes: relatively low optical density coupled with high visual contrast, freedom from metallic "mirroring," low background staining of subjacent muscle fibers, and presentation of a polychromatic picture. The entire procedure was under the complete and repeatable control of the operator. Perikaryon and nuclear morphology were clearly demonstrated. The limitations of this method are that it does not provide good visualization of individual unmyelinated neuronal processes and does not permit preparation of permanent slides.

Morphological and morphometric studies of cerebellar fissuration and myelination in mice with trisomy 19.

The fissuration and myelination in cerebella of trisomy 19 mice and of chromosomally balanced control animals on postnatal days (PD) 2, 5, 10, 12, and 15 were analyzed morphologically and morphometrically. In trisomy 19 mice, an increasing fissuration lag with a fissuration pattern and sequence identical to those of the control animals was found. The nuclear volume density of the trisomic glia cell in the neocerebellar commissure also developed like that of control mice, with an increase shortly before and parallel to the beginning of myelination and with a decrease in its further course. The trisomic axons and myelin sheaths were normally structured but axonal growth and myelination in the neocerebellar commissure were retarded. The quantitative relations of the assumed axon/glia interactions were similar to those of the control animals. The fissuration and myelination in the cerebellum of trisomy 19 mice did not seem to be pathologically changed but seemed to be differently retarded. On PD 15, a myelination lag of less than 5 days but a fissuration lag of nearly 10 days was found. This observation and the assumption of a differently retarded growth of the cerebellar cortex compared with cerebellar subcortical regions lead to the suggestion that the trisomy might result in a progressively asynchronous and disproportional development of different CNS structures.

Unfolding and flattening the cortex of gyrencephalic brains.

The present paper describes a procedure for unfolding and flattening gyrencephalic brains that makes it possible to cut single tangential sections through extensive regions of originally convoluted cortex. This procedure involves incising the arachnoid, removing the white matter, and then opening up the gyri and sulci in tissue that has been fixed in such a way as to maintain much of its natural flexibility. A technique is also described for preserving the complete gyral pattern of the intact brain on the surface of a flattened cerebral hemisphere. Finally, examples are presented in which the gyral pattern in the cat brain is related to the location of identified cortical areas in myelin-stained tangential sections from flattened material.

A new approach toward analyzing peripheral nerve fiber populations. I. Variance in sheath thickness corresponds to different geometric proportions of the internodes.

The thickness of the myelin sheath is known to increase with axon caliber, but there is also a superimposed, slight variation in sheath thickness depending on whether a fiber of a given caliber has very long or very short internodes. This relationship between myelin sheath thickness and the geometric proportion of the internode has been shown in subserial sections of isolated nerve fibers. It allows a prediction of sheath thickness from the quotient internode length/axon caliber, or conversely, a prediction of internode foreshortening from sheath thickness. We applied this new approach to the analysis of sciatic fiber populations of frogs, mice, rats and cats. The geometric proportions of these fibers were defined by the quotient internode length/fiber caliber. This quotient was compared with minor variation in sheath thickness as determined with a computer-assisted technique measuring large numbers of fibers in low-power electron micrographs. The method also calculated fiber shrinkage and recalculated all data for circular fiber profiles. The data obtained confirmed previous electron microscopic measurements showing that there is a slight reduction in sheath thickness when a fiber of a given caliber has relatively short internodes, and vice versa. A population of very thin, thinly myelinated fibers was also revealed. Sheath thickness and the geometric proportions of internodes in frogs differed markedly from those in mammals.

A new approach toward analyzing peripheral nerve fiber populations. II. Foreshortening of regenerated internodes corresponds to reduced sheath thickness.

The new approach used in this study is based on the concept that axon caliber is not the only factor affecting the thickness of the myelin sheath. It is necessary to consider the entire geometric proportions of the internode, since sheath thickness corresponds to the relationship between axon caliber and the length of the internode. This type of analysis was applied to the regenerated internodes in rat sciatic nerves. Survival periods of 4, 9, 18 and 36 weeks were studied after lesions had been placed in young adult rats. The data show significantly thinner sheaths for regenerated fibers as compared with normal nerves, consistent with previous observations. This reduction in sheath thickness, however, corresponded quantitatively to the degree of foreshortening of internodes in the regenerated nerves. An average reduction of 10 in the quotient internode length/fiber caliber corresponded to a reduction of about 0.015 in the relative thickness of the sheath (quotient axon diameter/fiber diameter). This means that regenerated myelin sheaths are not truly hypoplastic; rather, they are adapted to the reduced internode length, and have the same relationship found for normal fibers. In partially damaged nerves there was a clear distinction in terms of sheath thickness between regenerated fibers and undamaged fibers. Demonstration of this phenomenon by scatter diagrams opens new possibilities for the quantitative assessment of neuropathies.

Peripheral nerve autografts to the injured spinal cord of the rat: an experimental model for the study of spinal cord regeneration.

Regenerated central axons have been shown experimentally to penetrate in peripheral nerve segments transplanted into the spinal cord (SC). However, if the nerves are transplanted between the stumps of the transected SC regeneration is impaired by local cavitation and scarring. Our experiment was designed to study whether nerve grafts bridging a severe transverse SC lesion might provide to central regenerating axons a pathway to by-pass the lesion. To this purpose, 2 segments of autologous peripheral nerves were inserted through small dural openings into dorsal longitudinal myelotomies rostral and caudal to a transverse SC lesion in rats. Eighteen weeks after transplantation a large number of well myelinated fibres filled the grafted nerves. Only a few of these fibres, however, could be followed into the SC; they were located in the outer layers of the dorsal white matter. The problems regarding the origin and destination of these fibres are discussed.

Sympathetic postganglionic unmyelinated axons in the rat peripheral nervous system.

We determined the contribution made to the unmyelinated axon population of the rat peripheral nervous system by sympathetic paravertebral ganglion cells. Sympathectomy, achieved by administration of guanethidine to neonatal rats, led to atrophy of the sympathetic paravertebral ganglion chain, a 95% decrease in peripheral nerve norepinephrine, and loss of 20 to 26% of the unmyelinated axons in a cutaneous nerve (sural), a muscular nerve (nerve to soleus), and a mixed nerve (sciatic). These data indicate that up to a quarter of the total population of peripheral nerve unmyelinated axons are sympathetic ganglia-derived.

Unmyelinated axon subpopulations in the rat peripheral nervous system.

Using the neurotoxin capsaicin, we examined subpopulations of unmyelinated axons in mixed (sciatic), cutaneous (sural), and muscular (nerve to soleus) nerves. Administration of capsaicin to neonatal rats caused reduction of the sciatic nerve immunoreactive (IR)-substance P (by 45%) and IR-somatostatin (by 84%) contents. This correlated with a substantial reduction in unmyelinated axons in the sciatic and sural nerves (45% and 65%, respectively), although there was no significant decrease in unmyelinated axons in the nerve to soleus. In a parallel study, we have shown that sympathetic ganglia-derived unmyelinated axons account for about 20 to 25% of the total unmyelinated axon population in both the sural nerve and the nerve to soleus. Thus, in the sural nerve, the majority of unmyelinated axons are dorsal root ganglia-derived, contain either substance P or somatostatin, and are capsaicin-sensitive; whereas in the nerve to soleus, the majority of unmyelinated axons are dorsal root ganglia-derived but are insensitive to capsaicin and do not contain substance P or somatostatin. These latter unmyelinated axons presumably contain a yet to be defined neurotransmitter and may be the axons connecting with muscular ergoreceptors, a subpopulation of unmyelinated axons that are biochemically and functionally distinct from the unmyelinated axons of cutaneous nerves.

Internodal myelin volume and axon surface area. A relationship determining myelin thickness?

Internodes from normal, remyelinated and regenerated nerve fibres have been isolated from rat spinal roots and sciatic nerve. The internodes have been examined quantitatively by light and electron microscopy to determine their internodal length, myelin thickness, and the circumference and cross-sectional area of both the axons and fibre. Comparison of these measurements of the axon and myelin sheath has revealed a close relationship between the volume of myelin comprising the internode and the area over which the Schwann cell and axon are in close proximity, i.e. the surface area of the axolemma beneath the internodal myelin sheath. The same relationship described not only the internodes on normal nerve fibres, where internodal length is proportional to axon diameter, but also the short and thinly myelinated internodes formed in the adult animal on remyelinated and on regenerated axons. Examination of data presented by Berthold (1978) revealed that a closely similar relationship is also present in feline nerve fibres. In view of the constancy of the relationship between such different types of internode it is suggested that the regulation of myelin volume, and thereby of myelin thickness, may be mediated via the area of the axolemma or of the Schwann cell membrane beneath the myelin sheath.

Myelination of the human vagus nerve from 24 weeks postconceptional age to adolescence.

Significant changes in respiratory reflexes occur with maturation. The vagus nerve, the pathway for the Hering-Breuer and irritant-receptor reflexes, was studied quantitatively in 33 infants and 5 adolescents. In the infants, total myelinated vagus fibers increased linearly (r m0.682, p less than 0.001) with postconceptional age (PCA), and by 40 weeks after conception, total counts were comparable to those of adolescent group. Counts of total myelinated vagus fibers in 16 term infants (greater than 41 weeks PCA) were comparable to those in the adolescent group (p less than 0.40), whereas 17 preterm infants (less than 38 weeks PCA) showed significantly fewer total myelinated vagus fibers than term or adolescent groups (p less than 0.001). Smaller-diameter (less than 2 micrometer) myelinated vagus fibers depended upon PCA in the preterm group (p less than 0.005), but were independent of PCA in the term group (p less than 0.5). Preterm infants have a higher percentage of small to total myelinated vagus fibers than term infants (p less than 0.1).