The CNS-PNS transitional zone of the rat. Morphometric studies at cranial and spinal levels.

The transitional zone is that length of rootlet containing both central and peripheral nervous tissue. The CNS-PNS interface may be defined as the basal lamina covering the intricately interwoven layer of astrocyte processes which forms the CNS surface and which is pierced by axons passing between the CNS and PNS. Study of transitional zone development defines morphologically the growth, relative movement and interaction of central and peripheral nervous tissues as they establish their mutually exclusive territories on either side of the CNS-PNS boundary, and helps to explain the wide variations in the form of the mature transitional zone. Nerve rootlets at first consist of bundles of bare axons. These become segregated by matrices of fine Schwann cell processes peripherally and of astrocyte processes centrally. The latter may prevent Schwann cell invasion of the CNS. Astrocyte processes branch profusely and come to form the principal central nervous tissue component of the transitional zone. Developmental changes in the transitional zone vary markedly between nerves, reflecting differences in its final morphology. Widespread relative movements and migration of CNS and PNS tissues take place during development, so that the central-peripheral interface changes shape and position, commonly oscillating along the proximodistal axis of the rootlet. For example, developing cervical ventral rootlets contain a transient central tissue projection, while that of lumbar ventral rootlets and to a lesser extent that of cervical dorsal rootlets alternately increase and decrease in length. In the developing cochlear nerve, a central tissue projection is present before birth, but regresses somewhat before a marked outgrowth of central nervous tissue along the nerve takes place, which reaches into the modiolus during the first week postnatum. During development, some astrocytic tissue may even break off and migrate distally into the root, giving rise to one or more glial islands within it. During the period immediately preceding birth, Schwann cells come to be present in very large numbers in that part of the rootlet immediately distal to the CNS-PNS interface, the proximal rootlet segment. Here they form prominent sleeves or clusters of closely packed cells which intertwine with and encapsulate one another on the rootlet surface. Such Schwann cell overcrowding in the proximal rootlet segment could result in part from distal overgrowth of the rapidly expanding CNS around axon bundles, which might strip the Schwann cells distally off the bundle segments so engulfed.(ABSTRACT TRUNCATED AT 400 WORDS)

Axon-myelin relationships in rat cranial nerves III, IV, and VI: a morphometric study of large- and small-fibre classes.

The primary objectives of this study were to determine (1) if quantitative axon-myelin relationships are similar for large- and for small-fibre classes within individual nerves and (2) if the same axon-myelin relationships hold for equivalent fibre classes in closely similar nerves. The oculomotor, trochlear, and abducent nerves of the rat were examined since they each contain distinct large- and small-fibre classes and are similar in a wide range of anatomical and developmental respects. Accordingly, morphometric analyses of axon-myelin relationships were performed separately on large and small fibres of each of the three nerves. Within each nerve, the setting of the relationship between the two parameters was found to be different for the two fibre classes: Scatterplots relating sheath thickness to axon perimeter for large fibres were shifted upwards relative to those for small fibres. These differences were also reflected in the positions of the regression lines fitted to the plots and in the g-ratios. Significant differences were found between nerves in relation to their large fibres: Those of the abducent nerve had significantly thicker sheaths, those of the oculomotor nerve had significantly smaller axon perimeters, and the myelin sheath-axon perimeter relationship of the abducent nerve differed significantly from that of the other two. This study therefore shows that morphometric axon-myelin relationships may differ significantly between equivalent fibre classes of nerves that are closely similar in respect of morphological class, central origin, peripheral distribution, developmental environment, and function.

Myelin-axon relationships in the rat phrenic nerve: longitudinal variation and lateral asymmetry.

It is known that the myelin sheath thickness-axon perimeter relationship varies between peripheral nerves. This study examines the possibility that that relationship may vary between levels along a given nerve or between corresponding levels of the right and left examples of the same nerve. The relationship is examined for large and small fibre classes at well separated upper and lower intrathoracic levels in the rat phrenic nerve. The study shows that the myelin-axon relationship differs between levels along the same nerve bundle in the same (intrathoracic) environment. Thus, for a given increase in the perimeter of large axons, sheath thickness increases significantly more at lower than at upper levels. In addition, myelin sheath thickness shows a statistically significant lateral asymmetry in favour of the left side for the large fibre class at the upper thoracic level. The setting of the myelin sheath thickness-axon perimeter relationship also differs between the large and small fibre classes at each level examined. Large fibres have proportionately thicker sheaths than small fibres and this difference is reflected in the significantly smaller g-ratio of the former. Systematic differences in the setting of the myelin sheath thickness-axon perimeter relationship between large and small fibre classes may be a widely occurring phenomenon. It may be concluded that the myelin-axon relationship varies significantly both within and between nerves and also between fibre classes. Accordingly, morphometric studies of normal or pathological nerves should take into account possible consistent longitudinal variation or lateral asymmetry in fibre parameters and myelin-axon relationships within a given nerve bundle or fibre class, in order to avoid introducing systematic bias and to minimize variance between samples.

Myelin-axon relationships established by rat vagal Schwann cells deep to the brainstem surface.

The central-peripheral transitional zones of rat dorsolateral vagal rootlets are highly complex. Peripheral nervous tissue extends centrally for up to several hundred micrometers deep to the brainstem surface along these rootlets. In some instances this peripheral nervous tissue lacks continuity with the peripheral nervous system (PNS) and so forms an island within the central nervous system (CNS). In conformity with the resulting complexity of the CNS-PNS interface, segments of vagal axons lying deep to the brainstem surface are myelinated by one or more intercalated Schwann cells, contained in peripheral tissue insertions or islands, at either end of which they traverse an astroglial barrier. Intercalated Schwann cells are thus isolated from contact or contiguity with the Schwann cells of the PNS generally. They are short, having a mean internodal length of around 60% of that of the most proximal Schwann cells of the PNS proper, which lie immediately distal to the CNS-PNS interface and which are termed transitional Schwann cells. The thickness of the myelin sheaths produced by intercalated Schwann cells is intermediate between that of transitional Schwann cells and that of oligodendrocytes myelinating vagal axons of the same calibre distribution. This is not due to limited blood supply or to insufficient numbers of intercalated Schwann cells, the density of which is greater than that of transitional Schwann cells. These factors are unlikely to restrict expression of their myelinogenic potential. Nevertheless, the regression data show that the setting of the myelin-axon relationship differs significantly between the two categories of Schwann cell. Thus, the myelinogenic response of Schwann cells to stimuli emanating from the same axons may differ between levels along one and the same nerve bundle. Mean myelin periodicity was found to differ between sheaths produced by intercalated and by transitional Schwann cells.

Age changes in axon number along the cervical ventral spinal nerve roots in rats.

Axon counts were made at two standardised levels of C7 ventral spinal nerve roots from 46 female rats representing nine ages between birth and 500 days. The objective was to provide a definitive account of proximodistal changes in axon numbers and of age changes in axon numbers both during postnatal development and at several stages during maturity. At each age there is a proximodistal increase in the numbers of axons in all categories examined (myelinated, promyelin, transitional, and fetal) between levels midway along the subarachnoid course of the root and where it is apposed to but separate from the dorsal root ganglion. During maturation and throughout maturity axon totals change similarly at both levels: After a slight increase immediately postnatum, they decline sharply between 4 and 20 days due to a marked loss of unmyelinated axons. A gradual decline in myelinated axon numbers continues to 500 days. While these changes are occurring, axon numbers in all categories show a proximodistal increase throughout. The magnitude of this increase lessens with age for all but the transitional category due to a preferential decrease in numbers distally. Though these observations do not differentiate between axon branching and looping of sensory axons into the ventral root as a cause of the proximodistal increase in numbers, they tend to support the former. At each age during maturation axon proportions at proximal and distal levels correspond well for each animal, indicating that axon segregation proceeds at related rates within each root. Age changes in axon proportions within the transitional and fetal categories indicate that the postnatal stage of axon segregation results from axon loss, rather than Schwann cell proliferation.

The growth and myelination of central and peripheral segments of ventral motoneurone axons. A quantitative ultrastructural study.

This study compares the growth and myelination of those parts of cervical ventral motoneurone axons in the spinal cord (the intramedullary segments) and in the ventral roots of fetal and young rats (up to 21 days postnatal). The same fibre bundles are examined centrally and peripherally. Myelination begins centrally and peripherally at about birth. However, the peripheral segments of some fibres may begin to become myelinated before the central. Over the first 3 weeks after birth the minimum circumference of peripheral segments of myelinated axons remains relatively constant at 3 mum but that of central segments falls from 2.5 mum to just over 1 mum. Axons within the same fibre bundles tend to be thinner and less heavily myelinated centrally than peripherally. With ageing, axon circumference becomes more strongly correlated with sheath thickness. The thickness of the sheath surrounding an axon of a given circumference does not differ statistically from one age to another or between central and peripheral segments. Studies of myelin sheath growth rate show that in the early stages glial and Schwann cells vary independently of one another in the rates at which they add new turns to sheaths around central and peripheral segments of axons in the same bundles.

The maturation of the ventral root-spinal cord transitional zone. An ultrastructural study.

Initially, ventral motoneurone axons in the transitional zone are closely apposed to one another. They subsequently become progressively separated by astrocyte processes which grow into the axon bundles. These processes become progressively more numerous and project into the ventral rootlet as a glial dome. This disappears with maturation, the surface of the transitional zone becoming level with that of the surrounding cord. At first, a considerable length of the axon in and deep to, the transitional zone is covered only by astrocyte processes. The sleeve of oligodendrocytic cytoplasm myelinating the axon extends distally along it towards the cord surface, thus decreasing the length of axon covered by astrocyte processes. Concurrently, the Schwann cell myelinating the most proximal peripheral internode becomes invaginated into the cord over lengths of 50 micrometer or more. Finger-like processes stem from its central end and abut on the nodal axolemma, as in peripheral nodes. However, a few astrocyte processes remain closely applied to the nodal axolemma, even at maturity. In the adult, the attachment zone consists of closely packed invaginations, each containing the central end of a Schwann cell and its myelin sheath, presenting a honeycomb appearance.

The maturation of the ventral root-spinal cord transitional zone. Part 2. A quantitative ultrastructural study of the dynamics of its early development.

Stereological methods were used to calculate the proportions made up by the various tissue components of the transitional zone between C7 ventral spinal nerve rootlets and the spinal cord in Wistar albino rats at 1, 4 and 12 days after birth. The zone is about 20 micrometer deep at each age studied. Astrocyte processes form its major component, and are most densely packed in the middle and inner thirds of the zone throughout the period examined. The zone projects about 10 micrometer into the ventral rootlet shortly after birth but this projection has disappeared by 12 days. This is likely to be the result of a relative overgrowth of the cord tissue surrounding the attachment of the rootlet. In the outer third of the zone (adjacent to the ventral rootlet), Schwann cell perikarya are prominent features at 1 day, but are virtually absent at 4 days and subsequently. Schwann cell material is absent from the middle and inner thirds of the transitional zone at 4 days. Between 4 and 12 days, there is a relative invagination of Schwann cells into the deepest part of the zone. CNS-PNS transitional nodes are almost all located in the outer third of the zone at 4 days, but by 12 days have become evenly distributed throughout the zone.

Incidence of sarcocysts in skeletal muscles of horses.

The incidence of sarcocysts was examined in postural, propulsive and respiratory muscles from 74 horses ranging in age from mid-gestation to 14 years post-natal. Cryostat sections were stained for myosin adenosine triphosphatase (ATPase) at pH 9.5 and the type of muscle fibre containing sarcocysts was identified. Sarcocysts were found in muscles from three animals, all aged 1 year or more. Counts showed that they displayed no preference for any particular muscle. However, fibres with a high activity for myosin ATPase were preferentially colonized. Transverse sectional profiles of sarcocysts showed a wide variation in size, shape and wall thickness. Both the proportion of horses infected and the intensity of infection per animal were considerably lower than those reported in other studies.

The impact of neurotrophin-3 on the dorsal root transitional zone following injury.

STUDY DESIGN: Morphological and Stereological assessment of the dorsal root transitional zone (DRTZ) following complete crush injury, using light microscopy (LM) and transmission electron microscopy (TEM). OBJECTIVES: To assess the effect of exogenous neurotrophin-3 (NT-3) on the response of glial cells and axons to dorsal root damage. SETTING: Department of Anatomy, University College Cork, Ireland and Department of Physiology, UMDS, University of London, UK. METHODS: Cervical roots (C6-8) from rats which had undergone dorsal root crush axotomy 1 week earlier, in the presence (n=3) and absence (n=3) of NT-3, were processed for LM and TEM. RESULTS: Unmyelinated axon number and size was greater in the DRTZ proximal (Central Nervous System; CNS) and distal (Peripheral Nervous System; PNS) compartments of NT-3-treated tissue. NT-3 was associated with a reduced astrocytic response, an increase in the proportion of oligodendrocytic tissue and a possible inhibition or delay of microglial activation. Disrupted-myelin volume in the DRTZ PNS and CNS compartments of treated tissue was lower, than in control tissue. In the PNS compartment, NT-3 treatment increased phagocyte and blood vessel numbers. It decreased myelinating activity, as sheath thickness was significantly lower and may also account for the noted lower Schwann cell and organelle volume in the test group. CONCLUSIONS: Our observations suggest that NT-3 interacts with non-neuronal tissue to facilitate the regenerative effort of damaged axons. This may be as a consequence of a direct action or indirectly mediated by modulation of non-neuronal responses to injury.

Initial motor axon outgrowth from the developing central nervous system.

Rat and chick studies show that the earliest motor rootlet axon bundles emerge from all levels of the neural tube between radial glial end feet which comprise the presumptive glia limitans. The loose arrangement of the end feet at the time of emergence facilitates this passage. The points of emergence are regularly spaced in relation to the long axis of the neural tube and are not defined by any cell contact with its surface. Each rootlet carries a covering of basal lamina from the neural tube surface, which forms a sleeve around it. It is only after bundles of ventral rootlet axons have emerged that cells associate with them, forming clusters on the rootlet surface at a distance peripheral to the CNS surface of both species. A tight collar of glial end feet develops around the axon bundle at the neural tube surface shortly after initial emergence. These arrangements are in sharp contrast to those seen in the sensory rootlets, where clusters of boundary cap cells prefigure the sensory entry zones at the attachments of the prospective dorsal spinal and cranial sensory rootlets. Boundary cap cells resemble cluster cells and a neural crest origin seems the most likely for them. The study clearly demonstrates that no features resembling boundary caps are found in relation to the developing motor exit points.

Axon numbers in rat oculomotor, trochlear and abducent nerves.

In the rat oculomotor, trochlear and abducent nerves, large and small classes of myelinated fibres can be clearly distinguished. Small myelinated axons comprise a larger proportion of the total in the oculomotor nerve than in the other two. Mean counts enable the myelinated preganglionic parasympathetic outflow of the oculomotor nerve to be estimated at 216 fibres. Unmyelinated fibres are most frequent in the abducent nerve and least frequent in the trochlear nerve.

The transitional zone and CNS regeneration.

Most nerves are attached to the neuraxis by rootlets. The CNS-PNS transitional zone (TZ) is that length of rootlet containing both central and peripheral nervous tissue. The 2 tissues are separated by a very irregular but clearly defined interface, consisting of the surface of the astrocytic tissue comprising the central component of the TZ. Central to this, myelin sheaths are formed by oligodendrocytes and the supporting tissue is astrocytic. Peripheral to it, sheaths are formed by Schwann cells which are enveloped in endoneurium. The features of transitional nodes are a composite of those of central and peripheral type. The interface is penetrated only by axons. It is absent at first. It is formed by growth of processes into the axon bundle from glial cell bodies around its perimeter. These form a barrier across the bundle which fully segregates prospectively myelinated axons. Rat spinal dorsal root TZs have been used extensively to study CNS axon regeneration. The CNS part of the TZ responds to primary afferent axon degeneration and to regenerating axons in ways which constitute a satisfactory model of the gliotic tissue response which occurs in CNS lesions. It undergoes gliosis and the gliotic TZ tissue expands distally along the root. In mature animals axons can regenerate satisfactorily through the endoneurial tubes of the root but cease growth on reaching the gliotic tissue. The general objective of experimental studies is to achieve axon regeneration from the PNS through this outgrowth and into the dorsal spinal cord. Since immature tissue has a greater capacity for regeneration than that of the adult, one approach includes the transplantation of embryonic or fetal dorsal root ganglia into the locus of an extirpated adult ganglion. Axons grow centrally from the transplanted ganglion cells and some enter the cord. Other approaches include alteration of the TZ environment to facilitate axon regeneration, for example, by the application of tropic, trophic, or other molecular factors, and also by transplantation of cultured olfactory ensheathing cells (OECs) into the TZ region. OECs, by association with growing axons, facilitate their extensive regeneration into the cord. Unusually, ventral motoneuron axons may undergo some degree of unaided CNS regeneration. When interrupted in the spinal cord white matter, some grow out to the ventral rootlet TZ and thence distally in the PNS. The DRTZ is especially useful for quantitative studies on regeneration. Since the tissue is anisometric, individual parameters such as axon numbers, axon size and glial ensheathment can be readily measured and compared in the CNS and PNS environments, thereby yielding indices of regeneration across the interface for different sets of experimental conditions.

Axon-glial relationships in early CNS-PNS transitional zone development: an ultrastructural study.

The CNS-PNS transitional zone of rat cervical ventral rootlets develops in two stages: first, axon segregation, then transitional node formation. This ultrastructural study examines the former. Material was prepared by standard methods. Shortly after they grow out from the neural tube, ventral motoneuron axon bundles are extensively segregated by a matrix of fine processes forming a barrier across the rootlet, just distal to the cord surface. These processes arise from cell clusters on the rootlet surface. This barrier is prominent until the period around birth, when it is replaced by a second in which the axons are completely segregated from one another. The perikarya and processes forming this barrier resemble those of the first, but lie at or just below the cord surface. Thus, beginning at the earliest stage, a barrier crosses the axon bundle and segregates its axons before axon segregation is advanced either in the PNS or (especially) in the CNS. This may prevent central Schwann cell migration. Evidence is presented suggesting that the second barrier may arise through a relative proximal relocation of the first, as the cord grows radially. Near the cord surface, a complete, funnel-shaped sleeve of glial processes surrounds the axon bundle. This is continuous at the cord surface with the glia limitans. It constitutes an integral part of the transitional zone apparatus. It is also continuous centrally with the sheath which enfolds the bundle of ventral motoneuron axons as they run between the ventral horn and the transitional zone. Axon segregation at the cord surface, and therefore the formation of the definitive astrocytic CNS-PNS barrier occur relatively (and perhaps surprisingly) late at the cord surface. The definitive sharp discontinuity of central and peripheral tissue types characteristic of the transitional zone is established only after birth.

On methods of measuring nerve fibres.

Various methods of measuring axonal circumference and cross sectional area were examined. In measuring circumference, the Reichert MOP semi-automatic image analysis system varied more than a map meter. Several methods of area measurement were compared. Though all were relatively accurate in the long run, variability of individual measurements differed from one method to another, being greatest with a rolling disc planimeter and least with the method of counting millimetre squares within the profile to be measured. The map meter and the MOP were compared in measuring the circumferences of a large sample of axons: the latter method was significantly the more variable. Also, the MOP gave values significantly greater than the map meter. Comparison was also made of area measurements on the same large sample of axons, using various methods. Apart from the MOP and the rolling disc planimeter, each method gave area estimates significantly different from all others. The differences were in some cases as great as 4%. There was no significant correlation between the error involved and the size of the axon in measuring axonal circumference or area. Of all the methods studied, the MOP was the most efficient in measuring both circumference and area in terms of time taken and versatility. So long as a particular study is confined to comparing sets of measurements made by the same method, the systematic errors reported above are relatively unimportant. However, in comparing sets of measurements made with different methods, systematic errors may be important and should be borne in mind.

The ultrastructure of sheath cells in developing rat vomeronasal nerve.

Maturation of the vomeronasal nerve was studied in fetal, newborn and 3 months old rats. Early in development, each nerve consists of large numbers of bare axons with clusters of polygonal sheath cells lying at the periphery. The latter insinuate themselves between the axons which they segregate into bundles. The sheath cells and their processes which first delineate axon bundles from one another form a network in the interstices of which lie the emergent axon bundles. Each sheath cell is not confined to the sleeve around a single bundle. Its perikaryon and processes commonly contribute to the septa between several adjacent bundles. Eventually, each bundle comes to be surrounded by its own proper sheath which consists of processes of more than one Schwann cell. These developmental trends, of a progressive increase in the number both of axons per bundle and of Schwann cells associated with each bundle, are the reverse of those found in the PNS generally, where bundle size decreases and axon size increases with maturation. As individual bundles separate from one another, interfascicular collagen appears between them and each comes to be surrounded by a basal lamina. Separation is rarely complete, however; even at the mature stage, processes are exchanged between adjacent sheaths at one or more points on their circumferences. Schwann cell processes surrounding individual bundles become increasingly complex with maturation. Where adjoining processes meet, they commonly branch profusely and interdigitate intricately, forming stacks of closely apposed layered processes. In other areas, the branches are covered by basal lamina and bound intricate labyrinths which commonly extend deeply into the bundle and contain collagen fibrils.