Global expression of NGF promotes sympathetic axonal growth in CNS white matter but does not alter its parallel orientation.

Axonal regeneration is normally limited after injuries to CNS white matter. Infusion of neurotrophins has been successful in promoting regenerative growth through injured white matter but this growth generally fails to extend beyond the infusion site. These observations are consistent with a chemotropic effect of these factors on axonal growth and support the prevailing view that neurotrophin-induced axonal regeneration requires the use of gradients, i.e., gradually increasing neurotrophin levels along the target fiber tract. To examine the potential of global overexpression of neurotrophins to promote, and/or modify the orientation of, regenerative axonal growth within white matter, we grafted nerve growth factor (NGF) responsive neurons into the corpus callosum of transgenic mice overexpressing NGF throughout the CNS under control of the promoter for glial fibrillary acidic protein. One week later, glial fibrillary acidic protein and chondroitin sulfate proteoglycan immunoreactivity increased within injured white matter around the grafts. NGF levels were significantly higher in the brains of transgenic compared with non-transgenic mice and further elevated within injury sites compared with the homotypic region of the non-injured side. Although there was minimal outgrowth from neurons grafted into non-transgenic mice, extensive parallel axonal regeneration had occurred within the corpus callosum up to 1.5 mm beyond the astrogliotic scar (the site of maximum NGF expression) in transgenic mice. These results demonstrate that global overexpression of neurotrophins does not override the constraints limiting regenerative growth to parallel orientations and suggest that such factors need not be presented as positive gradients to promote axonal regeneration within white matter.

Re-establishment of neurochemical coding of preganglionic neurons innervating transplanted targets.

We investigated the effect on neurochemical phenotype of changing the targets innervated by sympathetic preganglionic neurons. In neonatal rats, the adrenal gland was transplanted into the neck, to replace the postganglionic neurons of the superior cervical ganglion. Transplanted adrenal glands survived, and contained noradrenergic and adrenergic chromaffin cells, and adrenal ganglion cells. Retrograde tracing from the transplants showed that they were innervated by preganglionic neurons that would normally have supplied postganglionic neurons of the superior cervical ganglion. The neurochemical phenotypes of preganglionic axons innervating transplanted chromaffin cells were compared with those innervating the normal adrenal medulla or superior cervical ganglion neurons. As in the normal adrenal gland, preganglionic nerve fibres apposing transplanted chromaffin cells were cholinergic. The peptide and calcium-binding protein content of preganglionic fibres was similar in normal and transplanted adrenal glands. In both cases, cholinergic fibres immunoreactive for enkephalin targeted adrenergic chromaffin cells, whilst cholinergic fibres with co-localised calretinin-immunoreactivity innervated noradrenergic chromaffin cells and adrenal ganglion cells. In contrast to the innervation of normal adrenal glands, these axons lacked immunoreactivity to nitric oxide synthase. In a set of control experiments, the superior cervical ganglion was subjected to preganglionic denervation in rat pups the same age as those that received adrenal transplants, and the ganglion was allowed to be re-innervated over the same time course as the adrenal transplants were studied. When the superior cervical ganglion was re-innervated by preganglionic nerve fibres, we observed that all aspects of chemical coding were restored, including cholinergic markers, nitric oxide synthase, enkephalin, calcitonin gene-related peptide and calcium binding proteins in predicted combinations, although the density of nerve fibres was always lower in re-innervated ganglia. These data show that the neurochemical phenotypes expressed by preganglionic neurons re-innervating adrenal chromaffin cells are selective and similar to those seen in the normal adrenal gland. Two explanations are advanced: either that contact of preganglionic axons with novel target cells has induced a switch in their neurochemical phenotypes, or that there has been target-selective reinnervation by pre-existing fibres of appropriate phenotype. Regardless of which of these alternatives is correct, the restoration of normal preganglionic codes to the superior cervical ganglion following denervation supports the idea that the target tissue influences the neurochemistry of innervating preganglionic neurons.

Release properties and functional integration of noradrenergic-rich tissue grafted to the denervated spinal cord of the adult rat.

Noradrenaline- (NA-) containing grafts of central (embryonic locus coeruleus, LC) or peripheral (juvenile adrenal medullary, AM, autologous superior cervical ganglionic, SCG) tissue were implanted unilaterally into rat lumbar spinal cord previously depleted of its NA content by 6-hydroxydopamine (6-OHDA) intraventricularly. A microdialysis probe was implanted in the spinal cord 3-4 months after transplantation, and extracellular levels of noradrenaline were monitored in freely moving animals during basal conditions and following administration of pharmacological or behavioural stimuli. Age-matched normal and lesioned animals both served as controls. Morphometric analyses were carried out on horizontal spinal sections processed for dopamine-beta-hydroxylase (DBH) immunocitochemistry, in order to assess lesion- or graft-induced changes in the density of spinal noradrenergic innervation, relative to the normal patterns. In lesioned animals, the entire spinal cord was virtually devoid of DBH-positive fibers, resulting in a dramatic 88% reduction in baseline NA, compared with that in controls, which did not change in response to the various stimuli. LC and SCG grafts reinstated approximately 80% and 50% of normal innervation density, respectively, but they differed strikingly in their release ability. Thus, LC grafts restored baseline NA levels up to 60% of those in controls, and responded with significantly increased NA release to KCl-induced depolarization, neuronal uptake blockade and handling. In contrast, very low NA levels and only poor and inconsistent responses to the various stimuli were observed in the SCG-grafted animals. In AM-grafted animals, spinal extracellular NA levels were restored up to 45% of those in controls, probably as a result of nonsynaptic, endocrine-like release, as grafted AM cells retained the chromaffine phenotype, showed no detectable fibre outgrowth and did not respond to any of the pharmacological or behavioural challenges. Thus, both a regulated, impulse-dependent, and a diffuse, paracrine-like, NA outflow may play roles in the recovery of lesion-induced sensory and/or motor impairments previously reported with these types of grafts following transplantation into the severed spinal cord.

Mature but not fetal or neonatal rat superior cervical ganglion transplants survive in the cortex of adult rats.

The transplantation of catecholaminergic tissues is a possible therapy for parkinsonism. Central nervous tissue is suitable for transplantation only in the immature stage, whereas peripheral nervous tissue can also be transplanted when mature. The present study compares the development of fetal (17-20 embryonic day, E17-20), neonatal (1-3 postnatal day, P1-3) and mature (5-6-week-old) rat superior cervical ganglia after transplantation into the cerebral cortex of adult rats. The mature transplants survived in greater proportion and preserved their structural characteristics, although a considerable proportion of the neurons died. The perinatal transplants only survived sporadically, decreased in size and the surviving remnants failed to display a structure comparable to the adult ganglion in situ. Thus, the use of adult donors is not only a possibility but a necessity when superior cervical ganglion (probably any ganglion) is transplanted. This principle is radically different from that seen in the case of central nervous tissues, and can be understood by the analysis of the time curves of cell proliferation and programmed cell death (apoptosis) observed during the perinatal development of sympathetic ganglia.

Reinnervation of denervated iris by transplanted sympathetic ganglia: effect of neuronal age.

Neuronal outgrowth in vivo is aggressive postnatally, but is diminished with increasing age. This may be attributable to intrinsic features of the neuron or its interaction with other components of the developing organism. The purpose of this study was to determine if there is an age-dependent reduction in the intrinsic ability of sympathetic neurons to initiate fiber outgrowth. Superior cervical ganglia from donor rats aged 3-4, 11-12, 27-28 and 45-46 days were removed and transplanted to the anterior chamber of the sympathectomized eye of host rats 85-89 days of age. Ganglia with host irides were removed at 3, 6 and 10 days post-transplant and whole mounts were analysed using catecholamine histofluorescence for maximum sympathetic fiber density, length and initial rate of outgrowth. Fluorescent fibers were present in host irides of donors of all ages and at all post-transplant times. However, maximum fiber density was less for the 3-4-day-old donor ganglia (e.g. 43-71% of 11-46-day-old donor ganglia at 600 microns, 10 days post-transplant). Maximum fiber length was also less in the youngest group (e.g. 35-49% of 11-46-day-old donor ganglia, 10 days post-transplant). Further, the initial rate of outgrowth was decreased for the 3-4-day-old donor ganglia (128 +/- 46 microns/day for the 3-4-day-old ganglia vs 253 +/- 48 microns/day for the 11-12-day-old ganglia, 307 +/- 35 microns/day for the 27-28-day-old ganglia and 260 +/- 22 microns/day for the 45-46-day-old ganglia).(ABSTRACT TRUNCATED AT 250 WORDS)