Garth M. Bray

Axonal regeneration and synapse formation in the superior colliculus by retinal ganglion cells in the adult rat

In adult rats, one optic nerve was transected and replaced by a 4 cm segment of autologous peripheral nerve (PN) that linked one eye and the superior colliculus (SC) along a predominantly extracranial course. Retrograde and orthograde studies with the tracers HRP or rhodamine-B- isothiocyanate (RITC), as well as immunocytochemical neuronal labels, indicated the following: (1) Regenerating axons from the axotomized retinal ganglion cells extended along the entire PN grafts, covering a distance nearly twice that of the normal retinotectal projection of intact rats. (2) Some of these axons penetrated the SC and formed terminal arborizations up to 500 microns from the end of the graft. (3) By electron microscopy, the arborizations of these regenerated axons in the SC were seen as small HRP-labeled axonal profiles that contacted neuronal processes in the SC; some of these contacts showed pre- and postsynaptic membrane specializations. These findings indicate that injured retinal ganglion cells in the adult rat are not only able to regrow lengthy axons, but may also form synapses in the SC.

Potential of Schwann cells from unmyelinated nerves to produce myelin: a quantitative ultrastructural and radiographic study

SummaryIn adult mice, most nerve fibres in the cervical sympathetic trunk (CST) are unmyelinated whereas a large proportion of sural nerve fibres are myelinated. This study of nerve grafts in syngeneic mice was designed to determine if Schwann cells originating from the unmyelinated CST would produce myelin when in contact with regenerating axons of the sural nerve. Quantitative microscopy of tritiated thymidine-labelled CST segments grafted to unlabelled sural nerve stumps revealed that, one month after grafting, previously unmyelinated grafts contained many myelinated fibres. By phase and electron microscope radioautography, nearly 40% of the myelin-producing cells in the reinnervated graft were shown to have originated in the unmyelinated CST. These findings indicate that Schwann cells originating from unmyelinated fibres are able to differentiate into myelin producing cells.

Multipotentiality of Schwann cells in cross-anastomosed and grafted myelinated and unmyelinated nerves: Quantitative microscopy and radioautography

Cross-anastomoses and autogenous grafts of unmyelinated and myelinated nerves were examined by electron microscopy and radioautography to determine if Schwann cells are multipotential with regard to their capacity to produce myelin or to assume the configuration seen in unmyelinated fibres. Two groups of adult white mice were studied. (A) In one group, the myelinated phrenic nerve and the unmyelinated cervical sympathetic trunk (CST) were cross-anastomosed in the neck. From 2 to 6 months after anastomosis, previously unmyelinated distal stumps contained many myelinated fibres while phrenic nerves joined to proximal CSTs became largely unmyelinated. Radioautography of distal stumps indicated that proliferation of Schwann cells occurred mainly in the first few days after anastomosis but was also present to a similar extent in isolated stumps. (B) In other mice, CSTs were grafted to the myelinated sural nerves in the leg. One month later, the unmyelinated CSTs became myelinated and there was no radioautographic indication of Schwann cell migration from the sural nerve stump to the CST grafts. Thus, Schwann cell proliferation in distal stumps is an early local response independent of axonal influence. At later stages, axons from the proximal stumps cause indigenous Schwann cells in distal stumps from the previously unmyelinated nerves to produce myelin while Schwann cells from the previously unmyelinated nerves to produce myelin while Schwann cells from the previously myelinated nerves become associated with unmyelinated fibres. Consequently, the regenerated distal nerve resembled the proximal stump. It is suggested that this change is possible because Schwann cells which divide after nerve injury reacquire the developmental multipotentiality which permits them to respond to aoxonal influences.

Axonal Elongation in Peripheral and Central Nervous System Transplants

Publisher Summary This chapter discusses axonal elongation in peripheral and central nervous system (PNS/CNS) transplants. The effective regeneration of injured nerve fibers depends upon several events: the survival of the neuronal perikarya; an outgrowth of new branches from the proximal stumps of the severed axons; elongation, ensheathment, and increase in the caliber of the growing axons; the reestablishment of quantitatively and qualitatively appropriate terminal contacts; and the loss of redundant axon branches. In the peripheral nervous system (PNS), this sequence of events can lead to the restoration of nerve fiber structure and function. In the adult mammalian CNS, by contrast, neither the initial outgrowths from interrupted axons nor the collateral sprouts from intact neurons elongate for more than a few millimeters. Although several mechanisms both intrinsic and extrinsic to nerve cells must contribute to the overall failure of CNS regeneration, certain experimental evidence suggests that differences in axonal elongation after damage to the CNS and PNS are strongly influenced by the characteristics of the microenvironment that surrounds the injured fibers. The chapter discusses the grafting of Schwann cells into peripheral nerve. It also describes the transplantation of neurons and target tissues.

Persistent retrograde labeling of adult rat retinal ganglion cells with the carbocyanine dye diI

To study the retrograde labeling of intact and axotomized retinal ganglion cells (RGCs) over long periods of time, we applied the carbocyanine dye diI to the superior colliculi (SC) and dorsal lateral geniculate nuclei (dLGN) in adult albino rats and examined the retinas by fluorescence microscopy after different periods of survival. Retrogradely labeled RGCs, which were observed in the retinas as early as 3 days after application of the dye, gradually increased in density so that by 7 days more than 80% of the RGCs were labeled and by 30 days diI-labeled cell densities were similar to those observed after short applications of other tracers. Using short-term retrograde labeling with fast blue (FB) as an independent marker of RGCs, it was determined that these neurons remained labeled with diI for periods of up to 9 months without apparent leakage of the tracer to other retinal cells. In addition, diI labeling persisted in the somata of more than 80% of axotomized RGCs whose contact with the source of label had been interrupted for 3 months. Thus, we propose that retrogradely transported diI is a useful label for quantitative studies of neuronal populations, even after axotomy.

Ensheathment and myelination of regenerating PNS fibres by transplanted optic nerve glia

Interactions between PNS axons and CNS glia were studied morphologically by transplanting optic nerves into peripheral nerves in groups of rats and mice. Four to 11 months after grafting, small numbers of axons from the peripheral nerves had penetrated the CNS grafts where they became ensheathed and myelinated by CNS glia. Glial protuberances observed at the CNS-PNS interfaces suggested that there had been an active glial response to innervation by PNS axons. These findings provide experimental evidence that denervated CNS glia can be reinnervated and form myelin.

Regenerated synapses persist in the superior colliculus after the regrowth of retinal ganglion cell axons

SummarySynapse formation by retinal ganglion cell axons was sought in the superior colliculus of four adult rats 16–18 months after the optic nerve was transected and replaced by a peripheral nerve graft that guided regenerating RGC axons from the eye to the superior colliculus. The terminals of retinal ganglion cell axons were labelled by intravitreal injections of tritiated amino acids and studied by light and electron microscopic autoradiography. We found that (i) retinal ganglion cell axons had extended from the tips of the peripheral nerve grafts into the superior colliculus for approximately 350 μ,m; (ii) within the superior colliculus, some regenerated retinal ganglion cell axons became ensheathed by CNS myelin; (iii) retinal ganglion cell terminals formed asymmetric synapses with dendrites of neurons in the superficial layers of the superior colliculus, mainly the stratum griseum superficialis.Regenerated (n=418) and normal retinal ganglion cell terminals (n=1775) in the superior colliculus were compared in terms of their size (area, perimeter, and maximum diameter), contacts per terminal, contacts per 10 μm terminal perimeter, and post-synaptic structure contacted (dendritic spine, shaft, or soma). No statistically significant differences in the ultrastructural characteristics of the pre-synaptic profiles were apparent between the two groups. The post-synaptic structures contacted by axon terminals were similar in regenerated and control animals, although there were quantitative differences in the distributions of these contacts among dendritic spines and shafts.These results suggest that the regeneration of retinal ganglion cell axons in adult rats can lead to the formation of ultrastructurally normal synapses in the appropriate layers of the superior colliculus. The re-formed connections appear to persist for the life-span of these animals.

Axonal regeneration from GABAergic neurons in the adult rat thalamus

SummaryPeripheral nerve grafts were inserted into the thalamus in 27 Sprague-Dawley rats. From 6 weeks to 15 months later, horseradish peroxidase (HRP) was applied to the extracranial end of each graft and sections of the brains reacted for peroxidase histochemistry. Of the thalamic neurons that were retrogradely labelled with HRP, more than 80% were located in the reticular nucleus of the thalamus (RNT), a distinct group of nerve cells that contain glutamic acid decarboxylase (GAD)-like immunoreactivity and are presumably GABAergic. By combining immunocytochemistry with HRP histochemistry, it was possible to confirm that the RNT neurons that had grown axons into the peripheral nerves grafts retained their GAD-like immunoreactivity. The apparent selectivity in their regenerative responses of RNT neurons to peripheral nerve grafts may relate to special properties of the neurons that did and did not grow into the grafts.

Prolonged administration of NT-4:5 fails to rescue most axotomized retinal ganglion cells in adult rats

The survival of axotomized RGCs was increased by intravitreal NT-4/5 given by repeated injections or osmotic minipumps, but the effects were less complete than predicted. Compared to a single injection of the neurotrophin on day 0, second injections on days 3 or 7 only sustained an additional 10-20% of the RGCs on day 10. Minipumps augmented RGC survival up to 4-fold (50%) at 2 weeks but most RGCs were lost by 1 month. Thus, specific neurotrophins can rescue many RGCs soon after injury but long-term neuronal survival may require a better understanding of changes in neurotrophin receptors and interactions with other molecules.

Reactions of unmyelinated nerve fibers to injury. An ultrastructural study.

Abstract Reactions of unmyelinated nerves to injury were studied in the distal stumps of rabbit anterior mesenteric nerves following transection. These nerves, chosen because they are almost exclusively unmyelinated, were examined by phase contrast and electron microscopy at intervals from 12 h to 2 weeks after transection. Swollen axons containing mitochondria and other organelles were prominent in the proximal few mm of the distal stump of anterior mesenteric nerve trunks during the first 4 days after transection. As early as 6 days after injury, regenerative changes consisting of numerous small axons with an increased axon-Schwann cell ratio were observed; there was little trace of degenerating axons, or their debris. Thus the capacity of unmyelinated nerve fibers for rapid regeneration has been demonstrated. It is anticipated that this delineation of reactions in unmyelinated nerves will contribute to a greater understanding of functional and morphologic abnormalities in disorders of peripheral nerves.