Albert J. Aguayo

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.

Lengthy regrowth of cut axons from ganglion cells after peripheral nerve transplantation into the retina of adult rats

Ganglion cell axons regrew to approximately their normal length (2 cm) when autologous peripheral nerve segments were inserted into the retina of adult rats for 4-18 weeks. Retrograde labeling from the graft with HRP or combinations of two different fluorescent dyes applied to the optic tract and graft demonstrate that axons growing into the nerve transplants originated from axotomized ganglion cells rather than by sprouting of undamaged neurons. Axonal injury and graft proximity to neuronal somata appear as requisites for the elongation of these fibers.

Regeneration of long spinal axons in the rat

SummaryTo investigate regeneration of long spinal axons, the right lateral column of the rat spinal cord was cut at high cervical, low cervical, midthoracic or lumbar level, and one end of an autologous sciatic nerve segment was grafted to the spinal cord at the site of incision. Three to six months after operation, the origin of axons in the grafts was traced retrogradely with horseradish peroxidase injected into the grafts and, in some cases, anterogradely with radioautography of tritiated amino acids injected into the brainstem. Axons from each of the major lateral spinal tracts arising in the brainstem as well as axons ascending from the lower spinal cord succeeded in growing into low cervical grafts. However, long descending axons rarely regenerated after midthoracic or lumbar injury; axons ascending from lumbar segments of the spinal cord usually failed to enter high cervical grafts. Differences in axonal regrowth at the four segmental levels were not simply attributable to dwindling of axonal number in fibre tracts. Axonal regeneration from Clarkes column or the red nucleus was observed only with lesions causing atrophy of many neurons.There was no obvious example of a fibre tract in the lateral spinal columns from which axons failed to regenerate nor from which axons regenerated exceptionally well. Under the conditions of these experiments, the distance from cell body to injury appeared to be an important determinant of axonal regeneration.

Expression of the growth-associated protein GAP-43 in adult rat retinal ganglion cells following axon injury

We have studied the expression of the growth-associated protein GAP-43 after injury to the axons of adult rat retinal ganglion cells (CNS neurons that do not normally regenerate injured axons). Both the biosynthetic labeling of GAP-43 and the GAP-43 immunoreactivity of the retina increased after axotomy, but only when the injury was within 3 mm of the eye. These results suggest the following conclusions: First, axon injury is sufficient to alter GAP-43 expression in CNS neurons, even in the absence of regeneration. Second, mechanisms that regulate GAP-43 expression are sensitive to the length of uninterrupted axon remaining after injury. Finally, the conditions that favor increased GAP-43 are similar to those that favor regrowth of injured CNS axons into grafts of peripheral nerve, suggesting that GAP-43 induction is accompanied by an increased potential of injured CNS neurons to regenerate.

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.

Peripheral nerve autografts to the rat spinal cord: Studies with axonal tracing methods

In young adult female rats, autologous sciatic nerve segments were transplanted to the thoracic region of the spinal cord. The grafts became well innervated but led to no obvious functional improvement. The origin and termination of axons in the grafts was studied by retrograde neuronal labeling with horseradish peroxidase (HRP) and radioautographic axonal tracing. Studies with HRP indicated that some axons in the grafts originated from intrinsic CNS neurons with their cell bodies in nearby segments of the spinal cord and that others arose from dorsal root ganglia at the level of the grafts and at least 7 segments distal to them. After tritiated amino acids were injected into lumbar dorsal root ganglia, labeled axons could be followed into the grafts but not into the rostral spinal cord stumps. Together with other experimental observations, these results demonstrate a correlation between success or failure of elongation of dorsal root fibers and peripheral or central ensheathment at the axonal tip. The corticospinal tract was studied both with radioautography and retrograde axonal transport of HRP but no extension of its axons into peripheral nerve grafts was detected under these experimental conditions. The findings implicate both neuroglial and axonal factors in the feeble regenerative response usually seen after injury to the spinal cord.

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.

Transplantation of rat schwann cells grown in tissue culture into the mouse spinal cord

Injections of lysolecithin were used to produce acute focal demyelination in the dorsal columns of 2 strains of mice, the myelin mutant quaking and the normal C57BL/6J. A small collection of rat Schwann cells grown in tissue culture was transplanted with their collagen substrate into this demyelinated area. The host mice were immune-suppressed to prevent graft rejection. Evidence of remyelination by Schwann cells was seen in the dorsal columns from 2-18 weeks after implantation. Proof that these Schwann cells were foreign to the host was derived from their rejection after the recipient mice were allowed to recover immunological competence by discontinuation of the immune suppression and by transferring immune cells sensitized against the donor tissue. It was concluded that Schwann cells grown in vitro retain their potential to produce myelin when returned to an in vivo situation and can myelinate central axons of a xenogenic host.