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Notes: In neurodegenerative diseases, neurons undergo prolonged periods of sprouting. Whether this sprouting compromises these neurons is unknown. Here, we examined the effect of axotomy on adult motoneurons undergoing prolonged sprouting in transgenic mice that overexpress GAP-43 (growth-associated protein). Sciatic nerve injury in these adult mice results in motoneuron death, but has no effect in non-transgenic mice. Thus, continued growth of motor axons renders adult motoneurons susceptible to nerve injury and compromises their long-term survival. The progressive nature of neurodegenerative diseases may therefore be caused by prolonged sprouting.

Notes: The failure of some CNS neurons to up-regulate growth-associated genes following axotomy may contribute to their failure to regenerate axons. We have studied gene expression in rat corticospinal neurons following either proximal (intracortical) or distal (spinal) axotomy. Corticospinal neurons were retrogradely labelled with cholera toxin subunit B prior to intracortical lesions or concomitantly with spinal lesions. Alternate sections of forebrain were immunoreacted for cholera toxin subunit B or processed for mRNA in situ hybridization for ATF3, c-jun, GAP-43, CAP-23, SCG10, L1, CHL1 or krox-24, each of which has been associated with axotomy or axon regeneration in other neurons. Seven days after intracortical axotomy, ATF3, c-jun, GAP-43, SCG10, L1 and CHL1, but not CAP-23 or krox-24, were up-regulated by layer V pyramidal neurons, including identified corticospinal neurons. The maximum distance between the lesion and the neuronal cell bodies that up-regulated genes varied between 300 and 500 µm. However, distal axotomy failed to elicit changes in gene expression in corticospinal neurons. No change in expression of any molecule was seen in the neocortex 1 or 7 days after corticospinal axotomy in the cervical spinal cord. The expression of GAP-43, CAP-23, L1, CHL1 and SCG10 was confirmed to be unaltered after this type of injury in identified retrogradely labelled corticospinal neurons. Thus, while corticospinal neuronal cell bodies fail to respond to spinal axotomy, these cells behave like regeneration-competent neurons, up-regulating a wide range of growth-associated molecules if axotomized within the cerebral cortex.

Notes: Summary Grafts of optic nerve were placed end-toend with the proximal stumps of severed common peroneal nerves in inbred mice. It was found that fraying the proximal end of adult optic nerve grafts to disrupt the glia limitans increased their chances of being penetrated by regenerating peripheral nerve fibres. Suturing grafts to the proximal stump also enhanced their penetration by axons. The maximum distance to which the axons grew through the CNS tissue remained about 1.5 mm from 2–12 weeks after grafting. Schwann cells were seldom identified in the grafts. Varicose and degenerating nerve fibres were often seen within the grafts. Some varicose profiles were shown to be the terminal parts of axons within the grafts. Axons containing clusters of organelles resembling synaptic vesicles became more abundant in the longerterm grafts. Immunohistochemical studies performed on sutured grafts using a polyclonal antiserum to neurofilaments confirmed the impressions given by the electron microscopical observations. Grafts of neonatal optic nerve lacked myelin debris but were not usually penetrated by regenerating peripheral axons within a 6-week period. Sixty minutes after the intravenous injection of horseradish peroxidase, reaction product could be detected in the extracellular spaces around blood vessels in all types of living optic nerve graft. This indicates that blood-borne macromolecules could penetrate the grafts. However the profiles of axons which were found within living optic nerve grafts had no obvious relationship to blood vessels and were usually surrounded by astrocytic processes. These results suggest that living astrocytes, rather than the absence of serum-derived trophic factors or the presence of CNS myelin, constitute the major barrier to the extension of axons and the migration of Schwann cells into CNS tissue.

Notes: Summary Freeze-dried tibial nerve grafts were anastomosed to either the proximal stump or the distal stump of severed tibial nerves in adult inbred Fischer rats. In the case of grafts attached to the proximal stump the tibial nerve was ligated three times, the most distal ligature from the spinal cord being 1 cm from the site of anastomosis. In both types of experiment Schwann cells were, therefore, free to enter the initially acellular grafts without accompanying axons. The grafts were examined 17 days to 12 weeks after operation. Immunofluorescence for S-100 protein was used to evaluate the distance migrated by the Schwann cells and electron microscopy was used to examine the morphology of the cells which invaded the grafts. Schwann cell migration was similar from the proximal and distal stumps. The migrating Schwann cells formed columns which resembled bands of Bungner. They were found mainly, but not exclusively, inside the pre-existing basal lamina tubes left behind by the killed nerve fibres. Some Schwann cells secreted a thin, patchy basal lamina even though they lacked axonal contact. Schwann cell columns became partially compartmentalized by fibroblast processes. Myelin and other debris were removed most rapidly in those parts of the grafts penetrated by large numbers of Schwann cells. The maximum distance the Schwann cells penetrated into the grafts was 8.5 mm and this was achieved by 6 to 8 weeks after operation. This is about half the maximum distance migrated by Schwann cells accompanying regenerating axons through similar grafts. The reasons why Schwann cells migrate shorter distances without axons and the significance of these results for the interpretation of axonal regeneration experiments using acellular grafts are discussed.

Notes: Summary A cavity was prepared in the rat parietal cortex by suction, filled with gel foam and left for 3 weeks during which time it became highly vascularised. Into this 3-week-old capillary bed a 5 mm length of autologous common peroneal nerve was implanted. Animals were killed at various time intervals up to 7 months after implantation of the nerve segment. The ultrastructural features of the vascular bed before and after implantation of the nerve segment were compared. In the absence of a peripheral nerve implant no axons were found within the cavity. However, at 5 weeks after implantation numerous axonlike profiles and capillaries containing fenestrations were observed within the implant. Eight weeks after implantation of the peripheral nerve both myelinated and non-myelinated axons were observed within the implant and in the surrounding capillary bed. No obvious increase in the number of axons was observed with increasing time periods. To investigate the origin of the axons within the vascular bed and/or implant the fluorochrome true blue was injected into the cavity 7 months after implantation of the nerve. Three days later selected areas of the brain, the trigeminal, superior cervical and otic ganglia were examined for retrogradely labelled fluorescent cells. Labelled cells were found adjacent to the cavity and in the ipsilateral trigeminal and superior cervical ganglia. The significance of these results in relation to the enhancement of axonal regeneration from the damaged central nervous system (CNS) is discussed.

Notes: Summary Although mature mammalian CNS neurons do not normally regenerate axons after injury, it is well established that they will regrow axons over long distances into peripheral nerve implants. We have autografted segments of sciatic nerve into the brains of adult albino rats and have used light and electron microscopic immunocytochemistry to examine the distribution of the growth associated protein GAP-43 in and around the graft in the first two weeks following implantation. GAP-43 was present, 3–14 days after grafting, in small non-myelinated axonal sprouts in the brain parenchyma around the proximal tip of the graft. At 11–14 days after implantation similar sprouts within the graft itself were GAP-43 immunoreactive. The sprouts were either naked or associated with other cell processes (chiefly of Schwann cells; to a lesser extent of astrocytes). We also show that small numbers of neuronal perikarya around the tip of the graft become GAP-43 immunoreactive 11–14 days after implantation. Thus mature mammalian CNS neurons regenerating axons into a PNS graft display a marked increase in their content of GAP-43. In addition, we report that small plaques of GAP-43 reaction product are sometimes present on the plasma membranes of Schwann cells or astrocytes adjacent to immunoreactive axons, and that narrow sheet-like or filopodial processes of astrocytes, Schwann cells and possibly other non-neuronal cell types, may contain small amounts of GAP-43.

Notes: Summary If one end of a segment of peripheral nerve is inserted into the brain or spinal cord, neuronal perikarya in the vicinity of the graft tip can be labelled with retrogradely transported tracers applied to the distal end of the graft several weeks later, showing that CNS axons can regenerate into and along such grafts. We have used transmission EM to examine some of the cellular responses that underlie this regenerative phenomenon, particularly its early stages. Segments of autologous peroneal or tibial nerve were inserted vertically into the thalamus of anaesthetized adult albino rats. The distal end of the graft was left beneath the scalp. Between five days and two months later the animals were killed and the brains prepared for ultrastructural study. Semi-thin and thin sections through the graft and surrounding brain were examined at two levels 6–7 mm apart in all animals: close to the tip of the graft in the thalamus (proximal graft) and at the top of the cerebral cortex (distal graft). In another series of animals with similar grafts, horseradish peroxidase was applied to the distal end of the graft 24–48 h before death. Examination by LM of appropriately processed serial coronal sections of the brains from these animals confirmed that up to several hundred neurons were retrogradely labelled in the thalamus, particularly in the thalamic reticular nucleus. Between five and 14 days after grafting, large numbers of tiny (0.05–0.20 μm diameter) nonmyelinated axonal profiles, considered to be axonal sprouts, were observed by EM within the narrow zone of abnormal thalamic parenchyma bordering the graft. The sprouts were much more numerous (commonly in large fascicles), smoother surfaced, and more rounded than nonmyelinated axons further from the graft or in corresponding areas on the contralateral side of animals with implants or in normal animals. At longer post-graft survival times, the number of such axons in the parenchyma around the graft declined. At five days, some axonal sprouts had entered the junctional zone between the brain and the graft. By eight days there were many sprouts in the junctional zone and some had penetrated the proximal graft to lie between its basal lamina-enclosed columns of Schwann cells, macrophages and myelin debris. Within the brain, sprouts were in contact predominantly with other sprouts but also with all types of glial cell. Within the junctional zone and graft many sprouts showed no consistent, close associations with other cell processes, although some were in contact or adjacent to processes of astrocytes, Schwann cells or macrophages. There was no evidence to suggest that axonal sprouts grew along astrocytic extensions to reach the junctional zone and graft. At eight days many axons in the junctional zone and graft were in contact with Schwann cell processes. Such axons, particularly those in intimate contact with the Schwann cell, were larger than those which had not established contact. By 14 days, most axons in the proximal graft were surrounded by Schwann cell processes, predominantly in basal lamina-enclosed columns. Some axons were associated with astrocyte processes, either in basal lamina-enclosed columns containing only astrocyte processes and axons or in columns containing a mixture of astrocyte and Schwann cell processes. The astrocyte processes involved in such bundles were concentrated at the periphery of the proximal graft, were not seen in the distal graft and probably represent long finger-like extensions of the astrocytes which rapidly form a glia limitans at the interface between brain and graft. This glia limitans was partially constructed at five days, almost complete at 14 days and subsequently became progressively thicker and more complex. At one month the proximal graft had acquired many of the features of a regenerating peripheral nerve and axons were present in large numbers in the distal graft. However the axon-Schwann cell relationships were immature in many of the Schwann cell columns both proximally and distally at one month, and virtually no myelination was apparent. At two months there were numerous myelinated fibres both proximally and distally although there were larger numbers of nonmyelinated axons, many in immature relationship with associated Schwann cells. Thus the graft appears to offer not only support for axonal elongation but also for a substantial degree of maturation of at least some of the regenerating axons, although (as will be reported elsewhere), the regenerated nerve fibres began to regress after two months.

Notes: Summary Guinea-pig inferior mesenteric ganglia (IMG)/hypogastric nerve preparations were incubated in tissue culture media containing ethylene glycol-bis-(β-aminoethyl ether)N,N′tetra-acetic acid (EGTA) or colchicine and examined either by fluorescence microscopy or by transmission electron microscopy. Two millimolar EGTA inhibited the accumulation of noradrenaline (NA) fluophore proximal to a crush, but did not produce an increase in the fluorescent intensity of, or the number of dense-cored vesicles (DCVs) within the neuronal perikarya. Calcium ions, but not magnesium ions, were able to block this effect of EGTA. Preparations incubated in the presence of colchicine (2.5 μg/ml) showed a reduction in the amount of fluorescent material accumulating proximal to a crash, but an increase in both the fluorescent intensity and the number of DCVs within the neuronal perikarya. The suggestion that calcium ions are required for the synthesis of some part of the NA-containing vesicle rather than for their loading onto the axoplasmic transport mechanism is discussed.

Notes: Summary A preparation comprising the guinea-pig inferior mesenteric ganglion (IMG) and ligated hypogastric nerves was maintainedin vitro in a twin-chamber apparatus. Horseradish peroxidase (HRP), added to the ganglion compartment, was taken up via coated pinocytotic vesicles into the neuronal perikarya, and subsequently accumulated in many polymorphic cytoplasmic organelles, but it did not enter the Golgi complex. After 24 h incubation, HRP was also localized in membrane-bounded organelles in the nonmyelinated axons of the hypogastric nerves in the ligated nerve compartment. HRP-labelled organelles accumulated in swollen nonmyelinated axons immediately proximal to the ligation along with other organelles known to undergo fast axoplasmic transport. It is suggested that HRP taken up by the neuronal perikarya subsequently underwent fast axoplasmic transport without passing through the Golgi complex. The organelles in which HRP was transported in an orthograde direction were very similar to those in which the enzyme is known to be transported retrogradely in the same neuronal system. The advantages of thisin vitro system for studying the orthograde transport of exogenous protein are discussed.

Notes: Summary The uptake and retrograde transport of horseradish peroxidase (HRP) and horseradish peroxidase-poly-l-lysine conjugate (HRP-PL) were compared using a system comprising the guinea-pig inferior mesenteric ganglion (IMG) and ligated hypogastric nerves maintainedin vitro in a twin chamber apparatus. 0.5mg of HRP-PL applied to the ligated nerves produced stronger retrograde labelling of neurons within the IMG than did 10 mg of HRP. This may have been due to the greater uptake of HRP-PL in a vesicular form by the axons immediately proximal to the ligation. The possible roles of large rounded vesicles and elongated cisternae in retrograde axoplasmic transport are discussed.