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Previous studies indicate that distal stumps of transected rat peripheral nerves secrete ‘tropic’ factors which can attract/support axonal regeneration over distances of several mm in vivo. The present study was undertaken in order to determine if there is specificity of neurotropic interaction at the level of the nerve trunk. Proximal stumps of transected peroneal or tibial nerves were inserted into the single inlet end of Y-shaped Silastic implants and offered alternative ‘lures’ at the paired outlet ends (specifically, grafts of peroneal vs tibial distal stump tissue). Several weeks later, the overwhelming majority of preparations showed exclusive growth of nerve fibers in implant forks attached to ‘native’ (originally associated) nerve stumps. Inversion of the distal stump grafts (such that the proximal stump was facing an analogous native distal stump, but a different region of it) diminished the frequency and extent of native preference. Taken together, data suggest the possibility that there can be a specificity nerve regeneration at the level of the nerve trunk.

Proximal stumps of rat sciatic nerveswere attached to inlet ends of Y-shaped silastic implants and offered a ‘choice’ of growing toward an Elvax pellet containing homogenate from previously crushed optic nerve which had been exposed to saline or cytosine arabinofuranoside (AraC). Previous studies indicate that AraC administration inhibits reactive gliosis in crushed optic nerve. Preferential or exclusive growth of axons occured in implant forks attached to pellets containing saline- (vs drug-) exposed optic nerve homogenates. In contrast, inhibition of Schwann cell mitosis had no discernible effect on tropic factor production in distal stumps of transected sciatic nerves. Tropic activity of homogenates from cultures containing reactive-like astrocytes was nearly 4 times higher than homogenates not containing these cells. Results suggest a possible link between formation of hypertrophic reactive astrocytes and production of neurotropic factor.

We re-examined the hypothesis of Cajal3, later refuted by Weiss and Taylor20, that cells in distal stumps of transected peripheral nerves exert an attractive (tropic) effect on regenerating axons. This question was re-assessed in vivo using surgical materials and assay procedures not available to those workers. Proximal stumps of transected rat sciatic or cat peroneal nerves were inserted into the single inlet end of a hollow, Y-shaped Silastic implant. Regenerating axons were provided with alternative targets consisting of a vacant arm vs one occupied by a sciatic nerve graft (rat), or a tibial (Tout) vs peroneal (Pout) distal nerve stump (cat). In some cases Pout was rendered metabolically compromised relative to Tout by exposing the former to dry ice and inhibitors of DNA and RNA synthesis. At 4.5 or 6 weeks postoperatively, the number of regenerating axons in each fork of the implant was assessed by morphometric analysis (total number of non-myelinated and myelinated axons greater than 1 micron in diameter at 4.5 weeks, and total number of myelinated axons at 6 weeks postoperatively), or by quantification of an axonally transported label. Rat sciatic nerve fibers exclusively regenerated toward the nerve graft, suggesting the existence of a neurotropic lure. In cats, morphometric analysis revealed a 10-(4.5 week) and 6-fold (6 week) greater number of axons growing towards untreated Tout vs treated Pout. When both distal stumps were untreated, more axons were seen in forks leading to Pout. Analysis of transported label confirmed the preferential growth of axons towards untreated Tout vs treated Pout for both motor and sensory axons. In separate experiments, Nuclepore filters (0.2 microns, pore size) were inserted between distal nerve stumps and outlet ends of Silastic implants. Preferential regeneration toward untreated stumps was observed if the distance between proximal and distal nerve stumps was equal to but not greater than 4-5 mm. These results suggest that peripheral nerve fiber regeneration in vivo can be directed by cells in distal stumps of transected nerves, and that this effect can be mediated over distances of several millimeters via diffusible factors.

Severance of a peripheral nerve leads to a characteristic series of events in the distal stump, including the dissolution of axons and myelin and the proliferation of Schwann cells within their basal lamina. This study examines the relationship between the spatial-temporal pattern of the induction of the Schwann cell S phase, loss of the structural and functional properties of axolemma, and the clearance of myelin debris in the cat tibial nerve. Nerve transection stimulated a monophasic increase in [3H]thymidine incorporation that peaked at 4 days post-transection throughout an 80-mm length of distal stump. Light microscope autoradiography revealed prominent incorporation into Schwann cells of myelinated fibres. Treatment of distal stumps with mitomycin C at the time of nerve transection greatly retarded thymidine incorporation and clearance of myelin debris, but not the time course of axonal degeneration, decline in the synthesis of the major myelin glycoprotein, P0, or the onset of ovoid formation. Nerve transection also greatly reduced the specific uptake of [3H]saxitoxin (STX), a ligand which binds to voltage-sensitive sodium channels. Binding in the distal stump fell precipitously to 20% of the normal at 4 days post-transection, concurrent with the peak of thymidine incorporation. This low level of binding was maintained for periods of up to 70 days, demonstrating that some STX binds to structures other than axons in denervated distal stumps. Prior treatment with mitomycin C delayed the loss of specific STX binding. In conclusion, these studies suggest that: (a) Schwann cell DNA replication- and/or mitosis regulates other events during Wallerian degeneration, including myelin degeneration, catabolism of P0 and the clearance of sodium channels from nodal axolemma; (b) the decline in P0 synthesis and/or shift to synthesis of less extensively processed P0 is independent of the induction of Schwann cell S phase; and (c) Schwann cells enveloping myelinated axons enter S phase within a 24-h period throughout the entire 80-mm length of distal stump.

Previous studies suggest that distal stumps of transected peripheral nerves contain diffusible factors which can attract/support axonal regeneration over distances of several mm in vivo. The present experiments were undertaken to determine if this is so for distal regions of traumatized central (i.e., optic) nerves. Proximal stumps of transected rat sciatic nerves were inserted into the single inlet ends of 6 mm long Y-shaped Silastic implants. Alternative ‘lures’ were attached to the paired outlets, the ability of these lures to attract/support regeneration of nerve fibers in their associated forks assessed 3.5 weeks postoperatively. Exclusive or preferential growth of nerve fibers occurred in implant forks associated with optic nerve grafts, of Elvax pellets containing homogenate obtained from previously crushed (reactive) optic nerves. Grafts of tendon, as well as homogenate from unoperated optic nerve had no effect. Results suggest that, with repect to the assay used, degenerating optic nerve tissue contains factor(s) which can attract/support regenerating nerve fibers.

The ability of a growth-promoting extract derived from bovine retina was assessed for its ability to support nerve fiber regeneration from proximal stumps of transected sciatic nerves. Proximal stumps of transected rat peripheral nerves were inserted into the single inlet end of a 6 mm long Y-shaped Silastic implant. One of the paired outlets was attached to an Elvax pellet containing the retina-derived growth extract, and the other outlet to a pellet containing an equivalent amount of tissue extract-free Hank's balanced salt solution. At 3 weeks postoperatively, the number of axons and blood vessels in the midportion of implant forks was assessed. The extent of axonal regeneration and blood vessel formation induced were similar to each other and dose-dependent. Results indicate preferential and dose-dependent growth of axons toward pellets containing the retina-derived growth-promoting extract.

Microtubules and neurofilaments are the major components of nerve fiber axoplasm and are evenly distributed longitudinally. Rearrangement of this distribution can be induced by a number of chemicals, including 2,5-hexanedione (2,5-HD). Utilizing 2,5-HD, this study explores the earliest time points of observable rearrangement of the cytoskeletal elements. Intrafascicular injections into the sciatic nerve shows central accumulation of microtubules and peripheral relocation of neurofilaments within 1 min of injection, suggesting a direct effect of the toxin of these components.

This paper describes a surgical method of manipulating feline peripheral nerve regeneration to separate spatially the temporal sequence of events of axon-Schwann cell interaction during nerve fibre formation. The method allows regenerating axons from the peroneal nerve to reinnervate the distal stump of a axon- and myelin-free, Schwann cell-enriched, chronically denervated tibial nerve distal stump. Three zones can be morphologically delineated in the tibial nerve stump after three weeks of reinnervation: (1) a proximal myelinated zone, (2) a more distal, non-myelinated, axon-Schwann cell contact zone, and (3) a distal axon-free Schwann cell non-contact zone. The distal limit of the second zone can be determined accurately by the front of an axonally transported label. The large volume of available tissue makes this method suitable for interdisciplinary studies to elucidate the control of axon elongation, axon growth, and axon-Schwann cell inter-relationships.

This study examined the thesis that 2,5-dexanedione (2,5-HD) produces distal (dying-back) axonopathy by direct toxic action on nerve fibers. Single or repeated application of undiluted or 10% 2,5-HD to exposed rat sciatic nerves caused some fibres to develop focal axonal swellings filled with abnormally large numbers of 10 nm neurofilaments. Such changes occurred within four days of application and were especially prominent in equivalently treated nerve segments obtained from animals orally exposed to 0.5% 2,5-HD before surgery. Nerve fibers along the perimeter of tibial nerve fascicles exposed to 2,5-HD or 2,4-hexanedione (2,4-HD), a compound unable to produce systemic neuropathy, underwent non-specific breakdown and Schwann cell necrosis. Nerve fibres located in the centre of such fascicles only developed an hypertrophied paranuclear Schwann cell cytoplasm and did not proliferate intermediate filaments. Saline, hydrochloric acid and 1,6-hexanediol, a water-soluble hexacarbon also lacking systemic neurotoxic properties, produced no intra-fascicular changes when locally applied to the sciatic nerve. It is concluded (1) that 2,5-HD causes a giant axonal swellings by direct toxic action on the nerve fibre, (2) 2,5-HD does not induce a generalized disorder of cytoplasmic intermediate filaments and (3) primary Schwann cell changes produced by locally applied 2,5-HD or 2,4-HD are non-specific and unrelated to the formation of the giant axonal swellings.

Summary The axonal migration of RNA, the nucleoside uridine and its nucleotide derivatives (NS/NT) were compared in neonatal and young adult rat optic axons. Tritiated uridine was injected into right eyes of developing (1- or 4-day-old) and young adult (40-day-old) rats which were sacrificed at times after injection ranging from 6 h to 20 days. Right and left lateral geniculates were removed and assayed for trichloroacetic acid soluble (NS/NT) and RNA radioactivity. Left minus right geniculate (L-RLG) radioactivity was used as an index of axonally migrating radioactitity. Results showed that uridine and its phosphorylated derivatives were transported along both neonatal and young adult rat optic axons. Greater than 90% of right geniculate (blood-borne) TCA soluble radioactivity was metabolized to volatile substances (probably3H2O) by three days after injection, leaving approximately 3% of the neonatal and approximately 10% of the adult activity as [3H]NS/NT. In left geniculate fractions (containing transported material) approximately 15 % and 40% of total TCA soluble radioactivity was present as [3H]NS/NT in neonates and adults, respectively. Thus, axonal NS/NT appears to be relatively protected from degradation when compared with blood-borne NS/NT. The amount of L-RLG [3H]RNA in the neonates was 10 times higher than in young adults. Peaks of neonatal [3H]RNA occurred at 5 and 10 days after birth, whether injections were made at 1 or 4 days of age indicating that this [3H]RNA may be linked to developmental events. Gel electrophoretic analysis of neonatal geniculate RNA indicated that a small portion of the [3H]RNA in the first peak represented axonally transported 4S RNA. The remainder of the L-RLG [3H]RNA in the neonates was probably due to a rapid and efficient incorporation of axonally transported [3H]NS/NT into extraaxonal geniculate RNA. In contrast, little or no axonal RNA transport could be demonstrated in the young adults.

Previous studies in this laboratory indicate that diffusible factors from distal stumps of transected peripheral nerves exert a neurotropic effect on regenerating nerve fibers in vivo9. The present study was performed (a) to strengthen this hypothesis, and (b) to begin to determine the distribution and identity of putative neurotropic factors in peripheral nerves. Rat sciatic nerves were transected and inserted into the inlet end of a hollow 6 mm-long Y-shaped implant made of medical-grade Silastic tubing. To one of the outlet ends was attached an Elvax pellet impregnated with whole homogenate from a single sciatic nerve. The other outlet was attached to a pellet not containing homogenate. Homogenates assayed were obtained from distal stumps of transected nerves derived 14 days postoperatively or from unoperated sciatic nerves. After 3.5 weeks, nerve fiber bundles consisting of myelinated regenerating axons were only present in implant forks attached to pellets containing denervated nerve homogenate. This activity was heat- and trypsin-sensitive. These data (a) strengthen the hypothesis that diffusible factors from distal nerve stumps of transected nerves can support nerve fiber regeneration, (b) indicate that those factors are protein and/or protein dependent, and (c) suggest that unoperated peripheral nerve (at the levels assayed) either do not contain such factors or contain substances which inhibit such factors.

The biochemistry of gliotic CNS tissue was assessed by monotoring changes in de novo protein synthesis distal to site of crush in the rat optic nerve between 3 and 20 days post-operatively. Radioactivity profiles on 12% polyacrylamide SDS gels showed reproducible peaks associated with protein(s) with dissociated molecular weights of 57K, 51K, 42K, 40K, 37K and 23K. Differences between crush and control nerves were observed with respect to the latter two peaks. De novo synthesis of 23K protein (comigrant with myelin proteolipid protein) was evident in control but not crushed nerves. Synthesis of 37K protein (identity unknown) was evident at 7, 10 and (to a lesser extent) 20 days post-operatively in crushed nerves, but not in crushed nerves at 3 days post-operatively or in unoperated nerves at any time point. The appearance of the synthesis of the 37K protein coincides with a drop in the level of functional axolemma (assessed by [3H]saxitoxin binding) in crushed nerves from 78% to 36% of control levels between 3 and 7 days post-operatively.

Glial and Schwann cells undergo marked biochemical and morphological alterations following axonal injury. In the present experiments, the extent of enzyme activity associated with anaerobic (LDH, lactic dehydrogenase) vs aerobic (SDH, succinic dehydrogenase) respiration was assessed distal to the site of nerve fiber injury. Studies were performed in rat central (optic) and peripheral (sciatic) nerves at 2, 7 and 14 days postoperatively (d.p.o.). In sciatic nerves, LDH activity rose 3-fold in traumatized (vs unoperated control) nerve tissue between 2 and 7 d.p.o. and remained elevated at 14 d.p.o. SDH activity in traumatized nerve was equal to that in unoperated nerve at 7 d.p.o., but decreased at 14 d.p.o. LDH activity in optic nerve at 2 d.p.o. was equivalent to that in control nerve, but rose approximately two-fold by 7 d.p.o. However, unlike peripheral nerve, activity in traumatized optic nerve decreased to control levels at 14 d.p.o. SDH activity in traumatized optic nerve remained unchanged at any timepoint examined. Taken in concert, these data are consistent with the hypothesis that there is an overall shift in CNS glial and Schwann cell metabolism from aerobic to anaerobic respiration following nerve injury. Additional studies were performed to determine if this shift requires prior Schwann or glial cell mitosis. Administration of mitotic inhibitor (AraC, cytosine arabinofuranoside) inhibited post-traumatic elevations in LDH activity in optic, but not peripheral nerve. No significant effect of the drug on axonal degeneration (as assessed by saxitoxin binding) was observed.

Studies in this laboratory have indicated that taurine is axonally transported along goldfish optic nerves. In the present experiments the axonal transport of taurine was examined in neonatal and young adult rat optic axons. [35S]taurine was injected into the vitreous humor of right eyes of developing (1--15-day-old) or young adult (40-day-old) rats. At various times after injection ranging from 3 h to 7 days, right retinae and left and right geniculates were removed and assayed for radioactivity, left minus right lateral geniculate (L-RLG) radioactivity being used as an index of axonally transported [35S]taurine. Results indicated that taurine was rapidly transported along both neonatal and young optic axons, in contrast to other amino acids (i.e., leucine and proline) which are not axonally transported in this system. Significant developmental variations were seen in both L-RLG and right retinal [35S]taurine activity 24 h after injection. The amounts of L-RLG [35S]taurine corrected for retinal ganglion cell uptake in animals injected at 1,4,7 and 11 days after birth (prior to and during the major period of synaptogenesis in the geniculates) were 4.5, 3.1, 2.3 and 2.6 times higher, respectively, than those in the young adults. In contrast, the amount of corrected L-RLG [35S]taurine in animals injected at 15 days after birth (after synaptogenesis) were not significantly different from that in the young adult.

Glutamine synthetase (GS) was assessed distal to the site of axonal injury in traumatized rat central (optic) or peripheral (tibial) nerve prior to and during periods of reactive PNS and CNS glial cell hypertrophy. GS activity in crushed optic nerve remained equivalent to that in unoperated control tissue at 2 days postoperatively, but rose by 30% at 7 and 12 days after surgery. In contrast, GS activity in traumatized peripheral nerve was significantly lower than in unoperated controls at 7 and 12 days postoperatively. Administration of mitotic inhibitor (AraC, cytosine arabinofuranoside) prevented significant trauma-induced alterations in GS activity in optic nerves at 7, and in peripheral nerves at 7 and 12 days postoperatively. Results suggest significant alterations in glial/Schwann GS activity after nerve fiber injury and that these alterations can be delayed/prevented by administration of mitotic inhibitors.