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Exogenous myelin- or axolemma-enriched fractions were assessed for the ability to inhibit biochemical and morphological expressions of reactive gliosis in rat optic nerve. Elvax pellets containing exogenous myelin, axolemma, whole-brain homogenate, liver, or red cell extracts or no homogenate were inserted into a dural slit in distal regions of crushed optic nerve. Biochemical and morphological expressions of reactive gliosis were assessed at 7 or 14 days postoperatively. Post-traumatic elevations in lactic dehydrogenase activity normally seen at 7 days postoperatively were prevented by placement of Elvax pellets containing myelin or axolemmal fractions into the optic nerve. Morphological analyses indicated an inhibition of post-traumatic elevations in glial cell numbers, surface area, and nuclear size at the 14-day time point. Exposure of the axolemmal fraction to heat or trypsin inactivated its ability to modulate reactive gliotic changes. Myelin fractions were trypsin-sensitive, but not heat-sensitive. In contrast, Elvax pellets containing whole-brain tissue homogenates or liver and red cell membranes had no significant effects on post-traumatic glial changes relative to preparations in which homogenate-free pellets were used.

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.

The short term effects of electric fields applied exogenously to the contused rat spinal cord were studied in a blind experiment. A DC field using 3 microA was applied to the dorsal cord when the lesion was made, and rats were behaviorally tested weekly for up to 3 weeks on an inclined plane. The results show that the "cathode rostral" orientation group of animals performed statistically significantly better on the inclined plane than either the "anode rostral" or the "no current" group. Counts of axons in the dorsal funiculi rostral to the lesion in the "cathode rostral" group were statistically significantly greater than in other groups. The greater number of axons rostral to the lesion site coupled with the superior behavioral performance of the "cathode rostral" group suggest that this treatment may provide an environment suitable for axonal growth and can facilitate functional recovery in the damaged mammalian spinal cord.

Dibutylcyclic AMP (DiBucAMP) can induce astroblast-containing cultures to form cells which resemble reactive astrocytes observed in vivo. In the present study, myelin- or axolemmal-enriched fractions were assessed for the ability to inhibit (DiBucAMP)-stimulated reactive-like changes in astrocytic cultures. Addition of exogenous myelin- or axolemmal-enriched fractions to DiBcAMP-exposed cultures prevented drug-induced elevation of lactic dehydrogenase (LDH) 2 days after initial addition of the drug and moderated DiBucAMP-induced decreases in the enzyme's activity normally observed 5 days later. The proportion of reactive colonies (i.e., those in which more than 50% of cells are stellate-shape) was significantly lower in cultures exposed to the drug plus myelin or axolemma vs those exposed to DiBucAMP alone. The inhibitory factors in axolemmal fractions were heat sensitive (at 100 degrees C), whereas those in myelin were not. Both fractions were inactivated by trypsin. Whole brain homogenates had no effect on diBucAMP-stimulated changes in culture.

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 have shown that the application of electric fields to the damaged mammalian nervous system is efficacious in promoting regeneration of peripheral nervous system axons. The present experiments were undertaken to determine whether exogenously applied electric fields can induce regenerative responses in the damaged mammalian central nervous system (CNS). In these studies, chronically delivered direct current was applied to the optic nerve, orienting the cathode distal to the lesion site. Histologic analyses at 3 weeks revealed that the damaged optic nerve did exhibit a regenerative response following treatment. We believe that this represents the first report of regenerative effects on the mammalian visual system using chronic DC electric fields, and we suggest that such galvanic treatment may be used alone or as an adjunct to promoting regeneration of the injured mammalian central nervous system.

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.

Previous studies indicated that axons from proximal stumps of transected peripheral nerves “prefer” to grow through Silastic tubes attached to their native (originally associated) rather than foreign (not originally associated) distal stumps. We determined whether or not this specificity is expressed at the level of the neuromuscular junction. Proximal stumps of transected rat sciatic nerves (peroneal and tibial branches) were attached to single inlet ends of 6-mm-long, Y-shape Silastic implants. One outlet was attached to the distal peroneal and the other to the distal tibial stump. Ten weeks later, innervation of the anterior tibialis and interosseous muscles (normally innervated predominantly by peroneal and tibial nerve fibers, respectively) was assessed by measuring compound muscle action potential amplitudes and latencies that follow supramaximal peroneal and tibial nerve stimulation. Results showed higher amplitudes in anterior tibialis muscle, induced by “native” peroneal (vs. tibial) stimulation in four of five animals, and higher amplitudes in interosseous muscles after “native” tibial (vs. peroneal) stimulation in all cases examined. Preparations in which bridges between proximal and distal nerve stumps were bridged with unbranched tubes showed random patterns of muscle innervation. The results suggest that if allowed to express “specificity” at the level of nerve trunk transection, regenerating mammalian peripheral axons can grow into, and form functional connection with, native (vs. foreign) muscle groups. This finding has possible clinical significance.

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.

The efficacy of applied electric fields in promoting regeneration of the transected and frozen rat sciatic nerve was studied. Three groups were studied at 6, 12, and 18 days post-lesion; nerves treated with 1.5 microA of direct current where the cathode was oriented distal to the lesion, nerves treated with the anode distal to the lesion, or no current delivered to the nerve. Regeneration in this severely damaged mammalian peripheral nerve model was quantitated by counting fluorescent antibody profiles to neurofilament protein distal to the lesion site. The results indicated that only cathodal current, applied distal to the lesion, significantly increased the rate of regeneration compared to controls. Such results help to elucidate mechanisms by which electric fields can facilitate regeneration in the mammalian nervous system.

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.

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.

Schwann cells are involved in all types of degenerative disorders of the peripheral nervous system, whether the primary lesion involves demyelination and remyelination, axonal degeneration and regeneration, or neuronal loss. The sequence of cellular changes in PNS disorders has been studied by examining the response of Schwann cells to loss and acquisition of axonal contact during Wallerian degeneration and nerve-fiber regeneration in the distal stumps of transected cat tibial nerves. Myelinating Schwann cells in distal stumps respond to axon degeneration by discarding and/or degrading their myelin sheaths, undergoing mitosis and adopting a quiescent state in which myelin-specific protein synthesis is suppressed. Myelin repair is activated by reassociation with regenerating axons destined to become myelinated: Schwann cells respond by undergoing cell division, expressing myelin-specific protein synthesis and elaborating a myelin sheath within a newly formed tube of basal lamina. The nature of the signalling mechanism from neuron to Schwann cell is unknown, although the interaction of specific ligands on the external surfaces of axon and Schwann cell is an attractive hypothesis. Since generally available methods have been developed to obtain fractions enriched in either the axolemma of myelinated fibers or the plasmalemma of quiescent Schwann cells free of neuronal regulation, the putative interactive properties of these surface membranes can now be explored.

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.

Previous studies have indicated that the application of electrical fields to the contused rat spinal cord could result in a partial return of function within 3 weeks after injury/treatment. Whether similar functional recovery could be established after a delay in the treatment was investigated. Rat spinal cords were contused and allowed to recover untreated for 10 days. At that time, a stimulator delivering 3 microA of direct current was applied to the dorsal portion of the cord such that the cathode was oriented either rostral or caudal to the lesion. Inactive stimulators were also used as controls. Rats were then assessed behaviorally and clinically at weekly intervals up to 3 weeks on an inclined plane and then killed for histological assessments. The results indicate that both the "cathode rostral" and "anode rostral" groups performed statistically significantly better on the inclined plane than the "no current" group. The cathode rostral and anode rostral group animals also demonstrated superior motor abilities. The number of axons in the dorsal funiculi rostral to the lesion in the actively treated groups were also statistically significantly greater than no current controls. The cathode rostral group animals demonstrated a greater number of viable neuronal cell bodies near the site of the lesion. These data suggest that electric fields may facilitate functional recovery and regeneration in the mammalian spinal cord, even after a delay in such treatment.

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.