Slow transport of the cytoskeleton after axonal injury

The delivery of cytoskeletal proteins to the axon occurs by slow axonal transport. We examined how the rate of slow transport was altered after axonal injury. When retinal ganglion cell (RGC) axons regenerated through peripheral nerve grafts, an increase in the rate of slow transport occurred during regrowth of the injured axons. We compared these results to axonal injury in the optic nerve where no substantial regrowth occurs and found a completely different response. Slow transport was decreased approximately tenfold in rate in the proximal segment of crushed optic nerves. This decreased rate of slow transport was not induced immediately, but occurred about 1 week after injury. To explore whether a decrease in the rate of slow transport was induced when the regeneration of peripheral nerves was physically blocked, we examined slow transport in motor neurons after the sciatic nerve was transected and ligated. In this case, no change in the rate of the comigrating tubulin and neurofilament (NF) radioactive peaks were observed. We discuss how the changes in the rate of slow transport may reflect different neuronal responses to injury and speculate about the possible molecular changes in the expression of tubulin which may contribute to the observed changes. © 1992 John Wiley & Sons, Inc.     

Slow transport of the cytoskeleton after axonal injury.

The delivery of cytoskeletal proteins to the axon occurs by slow axonal transport. We examined how the rate of slow transport was altered after axonal injury. When retinal ganglion cell (RGC) axons regenerated through peripheral nerve grafts, an increase in the rate of slow transport occurred during regrowth of the injured axons. We compared these results to axonal injury in the optic nerve where no substantial regrowth occurs and found a completely different response. Slow transport was decreased approximately tenfold in rate in the proximal segment of crushed optic nerves. This decreased rate of slow transport was not induced immediately, but occurred about 1 week after injury. To explore whether a decrease in the rate of slow transport was induced when the regeneration of peripheral nerves was physically blocked, we examined slow transport in motor neurons after the sciatic nerve was transected and ligated. In this case, no change in the rate of the comigrating tubulin and neurofilament (NF) radioactive peaks were observed. We discuss how the changes in the rate of slow transport may reflect different neuronal responses to injury and speculate about the possible molecular changes in the expression of tubulin which may contribute to the observed changes.     

Low Intensity Repetitive Transcranial Magnetic Stimulation Does Not Induce Cell Survival or Regeneration in a Mouse Optic Nerve Crush Model

Low intensity repetitive Transcranial Magnetic Stimulation (LI-rTMS), a non-invasive form of brain stimulation, has been shown to induce structural and functional brain plasticity, including short distance axonal sprouting. However, the potential for LI-rTMS to promote axonal regeneration following neurotrauma has not been investigated. This study examined the effect of LI-rTMS on retinal ganglion cell (RGC) survival, axon regeneration and levels of BDNF in an optic nerve crush neurotrauma model. Adult C57Bl/6J mice received a unilateral intraorbital optic nerve crush. Mice received 10 minutes of sham (handling control without stimulation) (n=6) or LI-rTMS (n = 8) daily stimulation for 14 days to the operated eye. Immunohistochemistry was used to assess RGC survival (β-3 Tubulin) and axon regeneration across the injury (GAP43). Additionally, BDNF expression was quantified in a separate cohort by ELISA in the retina and optic nerve of injured (optic nerve crush) (sham n = 5, LI-rTMS n = 5) and non-injured mice (sham n = 5, LI-rTMS n = 5) that received daily stimulation as above for 7 days. Following 14 days of LI-rTMS there was no significant difference in mean RGC survival between sham and treated animals (p>0.05). Also, neither sham nor LI-rTMS animals showed GAP43 positive labelling in the optic nerve, indicating that regeneration did not occur. At 1 week, there was no significant difference in BDNF levels in the retina or optic nerves between sham and LI-rTMS in injured or non-injured mice (p>0.05). Although LI-rTMS has been shown to induce structural and molecular plasticity in the visual system and cerebellum, our results suggest LI-rTMS does not induce neuroprotection or regeneration following a complete optic nerve crush. These results help define the therapeutic capacity and limitations of LI-rTMS in the treatment of neurotrauma.     

Changes in Neurofilament and Microtubule Distribution following Focal Axon Compression

Although a number of cytoskeletal derangements have been described in the setting of traumatic axonal injury (TAI), little is known of early structural changes that may serve to initiate a cascade of further axonal degeneration. Recent work by the authors has examined conformational changes in cytoskeletal constituents of neuronal axons undergoing traumatic axonal injury (TAI) following focal compression through confocal imaging data taken in vitro and in situ. The present study uses electron microscopy to understand and quantify in vitro alterations in the ultrastructural composition of microtubules and neurofilaments within neuronal axons of rats following focal compression. Standard transmission electron microscopy processing methods are used to identify microtubules, while neurofilament identification is performed using antibody labeling through gold nanoparticles. The number, density, and spacing of microtubules and neurofilaments are quantified for specimens in sham Control and Crushed groups with fixation at <1min following load. Our results indicate that the axon caliber dependency known to exist for microtubule and neurofilament metrics extends to axons undergoing TAI, with the exception of neurofilament spacing, which appears to remain constant across all Crushed axon diameters. Confidence interval comparisons between Control and Crushed cytoskeletal measures suggests early changes in the neurofilament spatial distributions within axons undergoing TAI may precede microtubule changes in response to applied loads. This may serve as a trigger for further secondary damage to the axon, representing a key insight into the temporal aspects of cytoskeletal degeneration at the component level, and suggests the rapid removal of neurofilament sidearms as one possible mechanism.     

Tissue-level thresholds for axonal damage in an experimental model of central nervous system white matter injury

In vivo, tissue-level, mechanical thresholds for axonal injury were determined by comparing morphological injury and electrophysiological impairment to estimated tissue strain in an in vivo model of axonal injury. Axonal injury was produced by dynamically stretching the right optic nerve of an adult male guinea pig to one of seven levels of ocular displacement (Nlevel = 10; Ntotal = 70). Morphological injury was detected with neurofilament immunohistochemical staining (NF68, SM132). Simultaneously, functional injury was determined by the magnitude of the latency shift of the N35 peak of the visual evoked potentials (VEPs) recorded before and after stretch. A companion set of in situ experiments (Nlevel = 5) was used to determine the empirical relationship between the applied ocular displacement and the magnitude of optic nerve stretch. Logistic regression analysis, combined with sensitivity and specificity measures and receiver operating characteristic (ROC) curves were used to predict strain thresholds for axonal injury. From this analysis, we determined three Lagrangian strain-based thresholds for morphological damage to white matter. The liberal threshold, intended to minimize the detection of false positives, was a strain of 0.34, and the conservative threshold strain that minimized the false negative rate was 0.14. The optimal threshold strain criterion that balanced the specificity and sensitivity measures was 0.21. Similar comparisons for electrophysiological impairment produced liberal, conservative, and optimal strain thresholds of 0.28, 0.13, and 0.18, respectively. With these threshold data, it is now possible to predict more accurately the conditions that cause axonal injury in human white matter.     

Taurine and Zinc Modulate Outgrowth from Goldfish Retinal Explants

Taurine and zinc, highly concentrated in the retina, possess similar properties in this structure, such as neuro-protection, membrane stabilization, influencing regeneration, and modulating development, maybe by acting in parallel or as interacting agents. We previously demonstrated that there are some correlations between taurine and zinc levels in hippocampus, dentate gyrus and retina of the developing rat. In the present study we evaluate the possible effects of taurine and zinc on outgrowth from goldfish retinal explants. The optic nerve was crushed 10 days before plating and culturing retinal explants in Leibovitz medium with 10% fetal calf serum and gentamicin. Neurites were measured with SigmaScanPro after 5 days in culture. Taurine (HPLC) and zinc (ICP) concentrations were determined in the retina between 1 and 180 days after crushing the optic nerve. Zinc sulfate (0.01–100 μM), N,N, N′,N′-tetrakis (pyridylmethyl) ethylenediamine (TPEN, 0.1–5 nM) and diethylenetriamine penta-acetic acid (DTPA, 10–300 μM), intracellular and extracellular zinc chelators, respectively, were added to the medium. TPEN was also injected intraocular (0.1 nM). Combinations of them were added with taurine (1–16 mM). Taurine concentrations were elevated in the retina 72 h after the crush, but were normalized by 180 days, those of zinc increased at 24 h, preceding the increase of taurine. The axonal transport of [³H]taurine from the optic tectum to the retina was not affected in fish with or without crush of the optic nerve at early periods after the injection, indicating an increase of it post-lesion. Zinc sulfate produced a bell-shaped concentration dependency on in vitro outgrowth, with stimulation at 0.05 μM, and inhibition at higher levels, also increased the effect of 4 mM taurine at 0.02 μM, but diminished it at higher concentrations in the medium. TPEN decreased outgrowth at 1 nM, but not at 0.5 nM, although the simultaneous presence of 4 mM taurine and 0.5 nM TPEN decreased outgrowth respecting the stimulation by taurine alone. The intraocular administration of TPEN decreased outgrowth in vitro, an effect counteracted by the addition of 4 mM taurine to the culture medium. DTPA decreased outgrowth from 10 μM in the medium. The present results indicate that an optimal zinc concentration is necessary for outgrowth of goldfish retinal explants and that, in zinc deficient retina, taurine could stimulate outgrowth. In addition, the observations of variations in tissue concentrations and of the effects of intraocular administration of TPEN indicate that these effects could occur in vivo. Special issue dedicated to Dr. Simo S. Oja     

Cyclosporin-A treatment attenuates delayed cytoskeletal alterations and secondary axotomy following mild axonal stretch injury

Following central nervous system trauma, diffuse axonal injury and secondary axotomy result from a cascade of cellular alterations including cytoskeletal and mitochondrial disruption. We have examined the link between intracellular changes following mild/moderate axonal stretch injury and secondary axotomy in rat cortical neurons cultured to relative maturity (21 days in vitro). Axon bundles were transiently stretched to a strain level between 103% and 106% using controlled pressurized fluid. Double-immunohistochemical analysis of neurofilaments, neuronal spectrin, alpha-internexin, cytochrome-c, and ubiquitin was conducted at 24-, 48-, 72-, and 96-h postinjury. Stretch injury resulted in delayed cytoskeletal damage, maximal at 48-h postinjury. Accumulation of cytochrome-c and ubiquitin was also evident at 48 h following injury and colocalized to axonal regions of cytoskeletal disruption. Pretreatment of cultures with cyclosporin-A, an inhibitor of calcineurin and the mitochondrial membrane transitional pore, reduced the degree of cytoskeletal damage in stretch-injured axonal bundles. At 48-h postinjury, 20% of untreated cultures demonstrated secondary axotomy, whereas cyclosporin A-treated axon bundles remained intact. By 72-h postinjury, 50% of control preparations and 7% of cyclosporin A-treated axonal bundles had progressed to secondary axotomy, respectively. Statistical analyses demonstrated a significant (p < 0.05) reduction in secondary axotomy between treated and untreated cultures. In summary, these results suggest that cyclosporin-A reduces progressive cytoskeletal damage and secondary axotomy following transient axonal stretch injury in vitro.     

Postinjury Changes in Platelet-Derived Growth Factor-Like Activity in Fish and Rat Optic Nerves

The poor regenerative ability of the CNS of mammals has been attributed, at least in part, to the presence of mature oligodendrocytes, which have been shown to inhibit axonal growth. Proliferation of oligodendrocyte progenitor cells in the rat optic nerve during development, and thereby the timing of oligodendrocyte differentiation, has been shown to depend on a factor derived from type 1 astrocytes, later characterized as platelet-derived growth factor (PDGF). In the present study we examine whether injury to the optic nerve induces changes in the levels of PDGF in spontaneously regenerating systems, compared with nonregenerating systems. Soluble substances, derived from nonneuronal cells surrounding injured fish and rat optic nerves, were prepared and examined for the presence of PDGF immunoreactivity and biological mitogenic activity on PDGF-responsive cells. The results suggest that PDGF-like mitogenic activity and immunoreactivity are present in both fish and rat optic nerves. However, in the rat optic nerve PDGF levels increased after axonal injury, whereas in the fish optic nerve injury was accompanied by an apparent decrease in PDGF-like levels. The results are discussed with respect to the possible role of PDGF in regeneration.     

Selective solubilization of high-molecular-mass neurofilament subunit during nerve regeneration

A reduction in neurofilament (NF) protein synthesis and changes in their phosphorylation state are observed during nerve regeneration. To investigate how such metabolic changes are involved in the reorganization of the axonal cytoskeleton, we studied the injury-induced changes in the solubility and axonal transport of NF proteins as well as their phosphorylation states in the rat sciatic nerve. In the control nerve, 15-25% of high-molecular-mass NF subunit (NF-H) was recovered in the 1% Triton-soluble fraction when fractionated in the presence of phosphatase inhibitors. After a complete loss of NF proteins distal to the injury site (70-75 mm from the spinal cord) 1 week after injury, NF-H detected in the regenerating sprouts at 2 weeks or later exhibited higher solubility (>50%) and lower C-terminal phosphorylation level than NF-H in the control nerve. Solubility increase was also apparent with L-[35S]methionine-labeled NF-H that was in transit in the proximal axon at the time of injury. The low-molecular-mass subunit remained in the insoluble fraction in both the normal and the regenerating nerves, indicating that selective solubilization of NF-H rather than total filament disassembly occurs during regeneration.     

Phosphorylation of Proteins in Normal and Regenerating Goldfish Optic Nerve

Within 6 h after radiolabeled phosphate was injected into the eye of goldfish, labeled acid-soluble and acid-precipitable material began to appear in the optic nerve and subsequently also in the lobe of the optic tectum, to which the optic axons project. From the rate of appearance of the acid-precipitable material, a maximal velocity of axonal transport of 13-21 mm/day could be calculated, consistent with fast axonal transport group II. Examination of individual proteins by two-dimensional gel electrophoresis revealed that approximately 20 proteins were phosphorylated in normal and regenerating nerves. These ranged in molecular weight from approximately 18,000 to 180,000 and in pI from 4.4 to 6.9. Among them were several fast transported proteins, including protein 4, which is the equivalent of the growth-associated protein GAP-43. In addition, there was phosphorylation of some recognizable constituents of slow axonal transport, including alpha-tubulin, a neurofilament constituent (NF), and another intermediate filament protein characteristic of goldfish optic axons (ON2). At least some axonal proteins, therefore, may become phosphorylated as a result of the axonal transport of a phosphate carrier. Some of the proteins labeled by intraocular injection of 32P showed changes in phosphorylation during regeneration of the optic axons. By 3-4 weeks after an optic tract lesion, five proteins, including protein 4, showed a significant increase in labeling in the intact segment of nerve between the eye and the lesion, whereas at least four others (including ON2) showed a significant decrease. When local incorporation of radiolabeled phosphate into the nerve was examined by incubating nerve segments in 32P-containing medium, there was little or no labeling of the proteins that showed changes in phosphorylation during regeneration. Segments of either normal or regenerating nerves showed strong labeling of several other proteins, particularly a group ranging in molecular weight from 46,000 to 58,000 and in pI from 4.9 to 6.4. These proteins were presumably primarily of nonneuronal origin. Nevertheless, if degeneration of the axons had been caused by removal of the eye 1 week earlier, most of the labeling of these proteins was abolished. This suggests that phosphorylation of these proteins depends on the integrity of the optic axons.     

Neurofilament phosphorylation in development. A sign of axonal maturation?

Monoclonal antibodies to the 200K neurofilament (NF) protein selectively decorated axons in tissue sections. Dilution of the antibodies in phosphate buffer and digestion with phosphatase abolished the stain. With conventional monoclonal and polyclonal NF antibodies, i.e. antibodies decorating NF regardless of their location (axons, perikarya and dendrites), the staining was not affected by this treatment. With all antibodies, axon-specific and conventional, the staining was abolished by trypsin digestion. Subsequent digestion with phosphatase did not restore the staining. Compared with conventional NF antibodies, staining with axon-specific anti-NF 200K was a late phenomenon in chick embryo development. NF 200K immunoreactivity was first observed in peripheral nerves and in the anterior columns of the spinal cord on day 10. Sensory ganglia and optic nerve fibers were negative. With conventional NF antibodies these structures were stained on days 4 and 5, respectively. In the following days of development the study was confined to the retina, optic nerves, cranial peripheral nerves and sensory ganglia. Up to day 16, bundles of thin peripheral nerve fibers, strongly decorated by conventional NF antibodies, did not stain with anti-NF 200K in double labelling experiments. Nerve bundles emerging from the ganglia were also negative, although some thick nerve fibers within the ganglia were stained. NF 200K immunoreactivity was first observed on day 17 in the optic nerve and in the layer of optic nerve fibers. At this time, staining was confined to the bundle emerging from the temporal side of the retina. In newborn chicken, only few fibers stained with anti-NF 200K in the nasal bundle, while the temporal bundle was well stained. It is suggested that the NF 200K antibodies reacted with a phosphorylated epitope in the axon, and that NF phosphorylation is a late event in ontogenesis probably related to axonal maturation.     

Phospho-dependent association of neurofilament proteins with kinesin in situ

Recent studies demonstrate co-localization of kinesin with neurofilament (NF) subunits in culture and suggest that kinesin participates in NF subunit distribution. We sought to determine whether kinesin was also associated with NF subunits in situ. Axonal transport of NF subunits in mouse optic nerve was perturbed by the microtubule (MT)-depolymerizing drug vinblastine, indicating that NF transport was dependent upon MT dynamics. Kinesin co-precipitated during immunoprecipitation of NF subunits from optic nerve. The association of NFs and kinesin was regulated by NF phosphorylation, since (1) NF subunits bearing developmentally delayed phospho-epitopes did not co-purify in a microtubule motor preparation from CNS while less phosphorylated forms did; (2) subunits bearing these phospho-epitopes were selectively not co-precipitated with kinesin; and (3) phosphorylation under cell-free conditions diminished the association of NF subunits with kinesin. The nature and extent of this association was further examined by intravitreal injection of (35)S-methionine and monitoring NF subunit transport along optic axons. As previously described by several laboratories, the wave of NF subunits underwent a progressive broadening during continued transport. The front, but not the trail, of this broadening wave of NF subunits was co-precipitated with kinesin, indicating that (1) the fastest-moving NFs were associated with kinesin, and (2) that dissociation from kinesin may foster trailing of NF subunits during continued transport. These data suggest that kinesin participates in NF axonal transport either by directly translocating NFs and/or by linking NFs to transporting MTs. Both Triton-soluble as well as cytoskeleton-associated NF subunits were co-precipitated with kinesin; these data are considered in terms of the form(s) in which NF subunits undergo axonal transport.     

Mechanisms of inflammation in spinal cord injury

An increase in protease activity is a hallmark event of the secondary injury cascade following contusion SCI. Elevated levels of protease activity result in the degradation of cytoskeletal components and myelin proteins essential for cellular function and survival. We have shown that a member of the cathepsin protease family is affected by SCI. The excessive release and activity of cathepsin B, a fairly ubiquitous lysosomal cysteine protease, has been implicated in several pathologies including tumor metastasis and progression, arthritis and Alzheimer's disease. Thus, our goal was to characterize any SCI-induced changes in cathepsin B expression. Following a T12 laminectomy and a moderate contusion (NYU device), the gene and protein profiles of cathepsin B in rats were examined using real-time PCR and immunoblots, respectively. Both the contusion injured animals and the time-matched sham controls exhibited elevated pro-enzyme protein levels (37 kDa form) at the lesion site, with significant differences between the two groups at 48 h, 72 h and 7 days post-SCI. Furthermore, there was a surge in the active species of the protein with significant differences at 72 h and 7 days post-SCI for the 30 kDa form and at 48 h. and 7 days for the 25 kDa form. Real-time PCR revealed increases in cathepsin B mRNA levels following contusion SCI as early as 6 h postinjury. These data indicate that SCI causes an up-regulation of cathepsin gene expression and protein levels, and suggest that this protease may be involved in the secondary injury cascade perhaps for as long as 1 week postinjury.     

The C-Terminal Domains of NF-H and NF-M Subunits Maintain Axonal Neurofilament Content by Blocking Turnover of the Stationary Neurofilament Network

Newly synthesized neurofilaments or protofilaments are incorporated into a highly stable stationary cytoskeleton network as they are transported along axons. Although the heavily phosphorylated carboxyl-terminal tail domains of the heavy and medium neurofilament (NF) subunits have been proposed to contribute to this process and particularly to stability of this structure, their function is still obscure. Here we show in NF-H/M tail deletion [NF-(H/M)^tailΔ] mice that the deletion of both of these domains selectively lowers NF levels 3–6 fold along optic axons without altering either rates of subunit synthesis or the rate of slow axonal transport of NF. Pulse labeling studies carried out over 90 days revealed a significantly faster rate of disappearance of NF from the stationary NF network of optic axons in NF-(H/M)^tailΔ mice. Faster NF disappearance was accompanied by elevated levels of NF-L proteolytic fragments in NF-(H/M)^tailΔ axons. We conclude that NF-H and NF-M C-terminal domains do not normally regulate NF transport rates as previously proposed, but instead increase the proteolytic resistance of NF, thereby stabilizing the stationary neurofilament cytoskeleton along axons.     

Protein and DNA oxidation in spinal injury: neurofilaments--an oxidation target

This study measured the time courses of protein and DNA oxidation following spinal cord injury (SCI) in rats and characterized oxidative degradation of proteins. Protein carbonyl content-a marker of protein oxidation-significantly increased at 3-9 h postinjury and the ratio 8-hydroxy-2-deoxyguanosine/deoxyguanosine-an indicator of DNA oxidation-was significantly higher at 3-6 h postinjury in the injured cords than in the sham controls. This suggests that oxidative modification of proteins and DNA contributes to secondary damage in SCI. Densities of selected bands on coomassie-stained gels indicated that most proteins were degraded. Neurofilament protein (NFP) was particularly evaluated immunohistochemically; its light chain (NFP-68) was gradually degraded in nerve fibers, neuron bodies, and large dendrites following SCI. A mixture of Mn (III) tetrakis (4-benzoic acid) porphyrin (10 mg/kg)-a novel SOD mimetic-and nitro-L-arginine (1 mg/kg)-an inhibitor of nitric oxide synthase-injected intraperitoneally, increased NFP-68 immunoreactivity and the numbers of NFP-positive nerve fibers post-SCI, correlating NFP degradation in SCI to free radical-triggered oxidative damage for the first time. Therefore, blockage of protein and DNA oxidation in the secondary injury stage may improve long-term recovery-important information for development of the SCI therapies.     

Poster Sessions CP12: Neurotrauma & Injury. Characterization of cathepsin B expression following contusion spinal cord injury (SCI)

An increase in protease activity is a hallmark event of the secondary injury cascade following contusion SCI. Elevated levels of protease activity result in the degradation of cytoskeletal components and myelin proteins essential for cellular function and survival. We have shown that a member of the cathepsin protease family is affected by SCI. The excessive release and activity of cathepsin B, a fairly ubiquitous lysosomal cysteine protease, has been implicated in several pathologies including tumor metastasis and progression, arthritis and Alzheimer's disease. Thus, our goal was to characterize any SCI‐induced changes in cathepsin B expression. Following a T12 laminectomy and a moderate contusion (NYU device), the gene and protein profiles of cathepsin B in rats were examined using real‐time PCR and immunoblots, respectively. Both the contusion injured animals and the time‐matched sham controls exhibited elevated pro‐enzyme protein levels (37 kDa form) at the lesion site, with significant differences between the two groups at 48 h, 72 h and 7 days post‐SCI. Furthermore, there was a surge in the active species of the protein with significant differences at 72 h and 7 days post‐SCI for the 30 kDa form and at 48 h. and 7 days for the 25 kDa form. Real‐time PCR revealed increases in cathepsin B mRNA levels following contusion SCI as early as 6 h postinjury. These data indicate that SCI causes an up‐regulation of cathepsin gene expression and protein levels, and suggest that this protease may be involved in the secondary injury cascade perhaps for as long as 1 week postinjury.     

Early cellular signaling responses to axonal injury

Background

We have used optic nerve injury as a model to study early signaling events in neuronal tissue following axonal injury. Optic nerve injury results in the selective death of retinal ganglion cells (RGCs). The time course of cell death takes place over a period of days with the earliest detection of RGC death at about 48 hr post injury. We hypothesized that in the period immediately following axonal injury, there are changes in the soma that signal surrounding glia and neurons and that start programmed cell death. In the current study, we investigated early changes in cellular signaling and gene expression that occur within the first 6 hrs post optic nerve injury.

Results

We found evidence of cell to cell signaling within 30 min of axonal injury. We detected differences in phosphoproteins and gene expression within the 6 hrs time period. Activation of TNFα and glutamate receptors, two pathways that can initiate cell death, begins in RGCs within 6 hrs following axonal injury. Differential gene expression at 6 hrs post injury included genes involved in cytokine, neurotrophic factor signaling (Socs3) and apoptosis (Bax).

Conclusion

We interpret our studies to indicate that both neurons and glia in the retina have been signaled within 30 min after optic nerve injury. The signals are probably initiated by the RGC soma. In addition, signals activating cellular death pathways occur within 6 hrs of injury, which likely lead to RGC degeneration.     

Amyloid Precursor Protein Is Synthesized by Retinal Ganglion Cells, Rapidly Transported to the Optic Nerve Plasma Membrane and Nerve Terminals, and Metabolized

Abstract: We have investigated the synthesis, axonal transport, and processing of the β-amyloid precursor protein (APP) in in vivo rabbit retinal ganglion cells. These CNS neurons connect the retina to the brain via axons that comprise the optic nerve. APP is synthesized in retinal ganglion cells and is rapidly transported into the optic nerve in small transport vesicles. It is then transferred to the axonal plasma membrane, as well as to the nerve terminals and metabolized with a f_1/2 of less than 5 h. A significant accumulation of C-terminal amyloidogenic or nonamyloidogenic fragments is seen in the optic nerve 5 h after [³⁵S]- methionine, [³⁵S]cysteine injection, which disappears by 24 h. The major molecular mass species of APP in the optic nerve is ∼110 kDa, and is an APP isoform that does not contain a Kunitz protease inhibitor domain. Higher molecular mass species containing this sequence are seen mostly in the retina. A protease(s) that can potentially cleave APP to generate an amyloidogenic fragment is present in the same optic nerve membrane compartment as APP.     

Wallerian degeneration and axonal regeneration after sciatic nerve crush are altered in ICAM-1-deficient mice

The intercellular cell adhesion molecule-1 (ICAM-1) has been implicated in the recruitment of immune cells during inflammatory processes. Previous studies investigating its involvement in the process of Wallerian degeneration and focusing on its potential role in macrophage recruitement have come to controversial conclusions. To examine whether Wallerian degeneration is altered in the absence of ICAM-1, we have analyzed changes in the expression of axonal and Schwann cell markers following sciatic nerve crush in wildtype and ICAM-1-deficient mice. We report that the lack of ICAM-1 leads to impaired axonal degeneration and regeneration and to alterations in Schwann cell responses following sciatic nerve crush. Degradation of neurofilament protein, the collapse of axonal profiles, and the re-expression of neurofilament proteins are substantially delayed in the distal nerve segment of ICAM-1^-/- mice. In contrast, the degradation of myelin, as determined by immunostaining for myelin protein zero, is unaltered in the mutants. Upregulation of GAP-43 and p75 neurotrophin receptor (p75^NTR) expression, characteristic for Schwann cells dedifferentiating in response to nerve injury, is differentially altered in the mutant animals. These results indicate that ICAM-1 is essential for the normal progression of axonal degeneration and regeneration in distal segments of injured peripheral nerves.