John P. Fraher

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.

Neurotrophin-3-mediated regeneration and recovery of proprioception following dorsal rhizotomy.

Injured dorsal root axons fail to regenerate into the adult spinal cord, leading to permanent sensory loss. We investigated the ability of intrathecal neurotrophin-3 (NT3) to promote axonal regeneration across the dorsal root entry zone (DREZ) and functional recovery in adult rats. Quantitative electron microscopy showed robust penetration of CNS tissue by regenerating sensory axons treated with NT3 at 1 and 2 weeks postrhizotomy. Light and electron microscopical anterograde tracing experiments showed that these axons reentered appropriate and ectopic laminae of the dorsal horn, where they formed vesicle-filled synaptic buttons. Cord dorsum potential recordings confirmed that these were functional. In behavioral studies, NT3-treated (but not untreated or vehicle-treated) rats regained proprioception. Recovery depended on NT3-mediated sensory regeneration: preventing regeneration by root excision prevented recovery. NT3 treatment allows sensory axons to overcome inhibition present at the DREZ and may thus serve to promote functional recovery following dorsal root avulsions in humans.

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)

Thoracic paravertebral block using real-time ultrasound guidance.

BACKGROUND: We developed a technique for ultrasound-guided paravertebral block, which was subsequently applied in the clinical setting. METHODS: An initial cadaver study was used to develop a technique that was used in the clinical setting on patients undergoing breast cancer surgery. RESULTS: Paravertebral catheters were correctly placed in the cadaveric trial in 8 of 10 attempts. In the clinical study, all blocked patients (n = 9) had evidence of thoracic wall sensory block and analgesia postoperatively. CONCLUSIONS: Determined by anatomical dissection, we have described the ultrasound features of the thoracic paravertebral space and performed clinically successful ultrasound-guided paravertebral block.

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.

A strong myelin thickness‐axon size correlation emerges in developing nerves despite independent growth of both parameters

The axon determines whether or not it is myelinated by the Schwann cell. At maturity there is a positive correlation between sheath thickness and axon calibre. This correlation is initially very low or absent, but gradually strengthens during development. This increase could come about because the axon continuously controls Schwann cell myelinating activity, so that a given axon calibre is associated with a particular myelin sheath thickness, an interaction which would entail the Schwann cell continuously monitoring and responding to axon size. This seems unnecessarily complex. This theoretical study shows that the strong correlation between the 2 parameters within a given myelinated fibre population may come about in a much simpler way than outlined above. This is demonstrated by modelling the growth and myelination of a hypothetical population, utilising data from earlier studies on cervical ventral motoneuron axon development. The hypothesis tested shows that the only instructive interactions by the axon on the Schwann cell necessary for the strong correlation between the 2 parameters to emerge are for the initiation of myelination, its continuation and its termination. These could result from a single stimulus being switched on, persisting for a time and being switched off. Under this influence, the Schwann cell is assumed to proceed to form the myelin sheath at a constant rate which it itself inherently determines, in the absence of any quantitative influence exerted by the axon. This continues until the stimulus for myelination ceases to emanate from the axon. The validity of the hypothesis is demonstrated, because the resulting myelin‐axon relationships correspond closely to those observed during development.

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.

Relative growth and maturation of axon size and myelin thickness in the tibial nerve of the rat

SummaryMorphometric observations have been made on the medial plantar division of the tibial nerve (MPD) and on the motor branches of the tibial nerve to the calf muscles (MBC) in rats ranging in age from weaning (3 weeks) to 12 months. Axon size, assessed by measurements of circumference and cross-sectional area, increased rapidly until 3 months with further slight increases between 3 and 9 months and a slight fall between 9 and 12 months. Axon size distributions were unimodal throughout in the MPD but bimodal for the MBC except at 3 weeks. Distributions of myelin thickness were bimodal throughout for both nerves. Scatter plots of g ratios (axon diameter: total fibre diameter) confirmed the presence of two fibre populations: a group of small fibres with relatively thin myelin sheaths, and a group of larger fibres within which sheath thickness was relatively less on the larger than on the smaller axons. These two fibres populations were less easily separable in the MBC than in the MPD nerves. These results document morphometrically the normal growth changes in the rat tibial nerve and also provide control data for the analysis of the effects of experimental procedures on the growth and maturation of peripheral nerve fibres.

Axons and glial interfaces: ultrastructural studies

At most vertebrate nerve transitional zones (TZs) there is a glial barrier which is pierced by axons passing between the CNS and PNS. Myelinated axons traverse this in individual tunnels. The same is true of larger non‐myelinated axons. This holds widely among the vertebrates, for example, the large motor axons of the sea‐lamprey Petromyzon (which also possess TZ specializations not found in mammals). Smaller non‐myelinated axons traverse the TZ glial tunnels as fascicles and so the barriers are correspondingly less comprehensive for them. Accordingly, in nerves composed of non‐myelinated axons, such as the vomeronasal or the olfactory, a TZ barrier stretching across the nerve is effectively absent. The chordate Amphioxus differs from the vertebrates in lacking a TZ barrier throughout. Invertebrates also lack glial barriers at the TZs between ganglia and interconnecting nerve trunks. The glial barrier at the dorsal spinal root TZ (DRTZ) has considerable value for analysing protocols aimed at achieving CNS regeneration, because it provides a useful model of the gliotic reaction at sites of CNS injury. Also, it is especially amenable to morphometric analysis, and so enables objective quantification of different protocols. Being adjacent to the subarachnoid space, it is accessible for experimental intervention. The DRTZ was used to investigate the value of neurotrophin 3 (NT3) in promoting axon regeneration across the TZ barrier and into the CNS following dorsal root crush. It promoted extensive regeneration and vigorous non‐myelinated axonal ensheathment. On average, around 40% of regenerating axons grew across the interface, compared with virtually none in its absence. These may have traversed the interface through loci occupied by axons prior to degeneration. Many regenerating axons became myelinated, both centrally and peripherally.

Fat head: an analysis of head and neck insulation in the leatherback turtle (Dermochelys coriacea).

SUMMARY Adult leatherback turtles are gigantothermic/endothermic when foraging in cool temperate waters, maintaining a core body temperature within the main body cavity of ca. 25°C despite encountering surface temperatures of ca. 15°C and temperatures as low as 0.4°C during dives. Leatherbacks also eat very large quantities of cold, gelatinous prey (medusae and pyrosomas). We hypothesised that the head and neck of the leatherback would have structural features to minimise cephalic heat loss and limit cooling of the head and neck during food ingestion. By gross dissection and analytical computed tomography (validated by ground truthing dissection) of an embalmed specimen we confirmed this prediction. 21% of the head and neck was occupied by adipose tissue. This occurred as intracranial blubber, encapsulating the salt glands, medial portions of the eyeballs, plus the neurocranium and brain. The dorsal and lateral surfaces of the neck featured thick blubber pads whereas the carotid arteries and jugular veins were deeply buried in the neck and protected laterally by blubber. The oesophagus was surrounded by a thick sheath of adipose tissue whereas the oropharyngeal cavity had an adipose layer between it and the bony proportion of the palate, providing further ventral insulation for salt glands and neurocranium.