Axonal Repair Research Articles

The role of Nogo-A in axonal plasticity, regrowth and repair

Axonal damage leads to permanent deficits in the adult central nervous system (CNS) not only because of the weak intrinsic ability of adult neurons to activate their growth program but importantly also because of the presence of specific growth inhibitors in the CNS tissue and the environment of the damaged axons. The well-studied myelin-derived protein Nogo-A is involved in various cellular and molecular events contributing to the failure of CNS axons to regrow and reconnect after transection. Recent studies have shown that, by acting in a negative way on the cytoskeleton and on the growth program of axotomized neurons, Nogo-A exerts fast and chronic inhibitory effects on neurite outgrowth. On the other hand, the blockade of Nogo-A results in a marked enhancement of compensatory and regenerative axonal extension in vivo; this enhancement is often paralleled by significant functional recovery, for example, of locomotion or skilled forelimb reaching after spinal cord or stroke lesions in rats and monkeys. Surprisingly, the blockade of Nogo-A or its receptor NgR in the hippocampus has recently been demonstrated to enhance long-term potentiation. A role of Nogo-A in synaptic plasticity/stability might therefore represent an additional, new and important aspect of CNS circuit remodeling. Function-blocking anti-Nogo-A antibodies are currently being tested in a clinical trial for improved outcome after spinal cord injury.

Axonal integrity predicts cortical reorganisation following cervical injury

BackgroundTraumatic spinal cord injury (SCI) leads to disruption of axonal architecture and macroscopic tissue loss with impaired information flow between the brain and spinal cord—the presumed basis of ensuing clinical...

Astrocytic αVβ3 integrin inhibits neurite outgrowth and promotes retraction of neuronal processes by clustering Thy-1.

Thy-1 is a membrane glycoprotein suggested to stabilize or inhibit growth of neuronal processes. However, its precise function has remained obscure, because its endogenous ligand is unknown. We previously showed that Thy-1 binds directly to αVβ3 integrin in trans eliciting responses in astrocytes. Nonetheless, whether αVβ3 integrin might also serve as a Thy-1-ligand triggering a neuronal response has not been explored. Thus, utilizing primary neurons and a neuron-derived cell line CAD, Thy-1-mediated effects of αVβ3 integrin on growth and retraction of neuronal processes were tested. In astrocyte-neuron co-cultures, endogenous αVβ3 integrin restricted neurite outgrowth. Likewise, αVβ3-Fc was sufficient to suppress neurite extension in Thy-1(+), but not in Thy-1(−) CAD cells. In differentiating primary neurons exposed to αVβ3-Fc, fewer and shorter dendrites were detected. This effect was abolished by cleavage of Thy-1 from the neuronal surface using phosphoinositide-specific phospholipase C (PI-PLC). Moreover, αVβ3-Fc also induced retraction of already extended Thy-1(+)-axon-like neurites in differentiated CAD cells as well as of axonal terminals in differentiated primary neurons. Axonal retraction occurred when redistribution and clustering of Thy-1 molecules in the plasma membrane was induced by αVβ3 integrin. Binding of αVβ3-Fc was detected in Thy-1 clusters during axon retraction of primary neurons. Moreover, αVβ3-Fc-induced Thy-1 clustering correlated in time and space with redistribution and inactivation of Src kinase. Thus, our data indicates that αVβ3 integrin is a ligand for Thy-1 that upon binding not only restricts the growth of neurites, but also induces retraction of already existing processes by inducing Thy-1 clustering. We propose that these events participate in bi-directional astrocyte-neuron communication relevant to axonal repair after neuronal damage.

Extensive cell migration, axon regeneration, and improved function with polysialic acid‐modified Schwann cells after spinal cord injury

Schwann cell (SC) implantation after spinal cord injury (SCI) promotes axonal regeneration, remyelination repair, and functional recovery. Reparative efficacy, however, may be limited because of the inability of SCs to migrate outward from the lesion-implant site. Altering SC cell surface properties by overexpressing polysialic acid (PSA) has been shown to promote SC migration. In this study, a SCI contusion model was used to evaluate the migration, supraspinal axon growth support, and functional recovery associated with polysialyltransferase (PST)-overexpressing SCs [PST-green fluorescent protein (GFP) SCs] or controls (GFP SCs). Compared with GFP SCs, which remained confined to the injection site at the injury center, PST-GFP SCs migrated across the lesion:host cord interface for distances of up to 4.4 mm within adjacent host tissue. In addition, with PST-GFP SCs, there was extensive serotonergic and corticospinal axon in-growth within the implants that was limited in the GFP SC controls. The enhanced migration of PST-GFP SCs was accompanied by significant growth of these axons caudal to lesion. Animals receiving PST-GFP SCs exhibited improved functional outcome, both in the open-field and on the gridwalk test, beyond the modest improvements provided by GFP SC controls. This study for the first time demonstrates that a lack of migration by SCs may hinder their reparative benefits and that cell surface overexpression of PSA enhances the ability of implanted SCs to associate with and support the growth of corticospinal axons. These results provide further promise that PSA-modified SCs will be a potent reparative approach for SCI. © 2012 Wiley Periodicals, Inc.

Recovery of Axonal Transport after Partial Optic Nerve Damage Is Associated with Secondary Retinal Ganglion Cell Death In Vivo

Traumatic injury of the optic nerve leads to retrograde cell death of retinal ganglion cells (RGCs) but usually a certain percentage of neurons survive. It has been suggested that recovery of axonal transport is beneficial for survival. The present study was therefore performed to provide a synopsis of the temporal pattern of axonal transport decline/recovery and the viability of RGCs after optic nerve crush (ONC). Fluorescent dyes were injected into the superior colliculus to retrogradely label RGCs. Axonal transport kinetics into the RGCs was visualized with in vivo confocal neuroimaging (ICON) in uninjured rats and in rats which had mild or moderate ONC. Red fluorescent beads were injected on day 2 post-ONC and green beads on day 7. At 2 to 4 days post-ONC significant axonal transport was detected, but within 1 week the transport of the fluorescent beads was decreased. Interestingly, during post-ONC week 3 the axon transport slowly recovered. However, despite this recovery, retrograde cell death rate continued and was even increased in a "second wave" of cell death in those neurons that displayed axon transport recovery. After damage many surviving RGCs lose their axon transport, but after approximately 3 weeks, this transport recovers again, a sign of intrinsic axon repair. Contrary to the prediction, axon transport recovery is not associated with better cell survival but rather with a second wave of cell death. Thus, the accelerated cell death associated with recovery of axon transport suggests the existence of a late retrograde cell death signal.

Progesterone and low-dose vitamin D hormone treatment enhances sparing of memory following traumatic brain injury

Progesterone (PROG) has been shown to protect the brain from traumatic injury and is now in Phase III clinical trials. Our work shows that PROG's beneficial effects can be reduced in vitamin D hormone (VDH)-deficient subjects. VDH can modulate neuronal apoptosis, trophic factors, inflammation, oxidative stress, excitotoxicity, and myelin and axon repair. We investigated whether VDH combined with PROG could improve behavioral outcomes more than PROG alone in VDH-sufficient rats given bilateral contusions of the medial frontal cortex. PROG and different doses of VDH (1μg/kg, VDH1; 2.5μg/kg, VDH2; 5μg/kg, VDH3) were injected intraperitoneally 1h post-injury. Eight additional doses of PROG were given subcutaneously over 8days with tapering over the last 2days. Neurobehavioral tests, necrotic cavity, neuronal death and activation of astrocytes were evaluated 21days post-injury. We found that PROG and PROG+VDH preserve spatial memory processing. VDH1+PROG improved performance in acquisition more effectively than PROG alone, indicating that the low VDH dose is optimal for combination therapy. There were no significant differences in necrotic cavity size among the groups. The density of positive staining for reactive astrocytes (glial fibrillary acidic protein (GFAP)) increased and the cell bodies and processes of GFAP-positive cells were enlarged in the PROG+VDH1 group. Our data indicate that the combination of PROG and VDH is more effective than PROG alone in preserving spatial and reference memory, and that PROG plus low-dose VDH can activate GFAP reactions up to 21days after injury. This effect may be one of the mechanisms underlying PROG's neuroprotective effects in combination with VDH.

Cellular mechanisms of plasmalemmal sealing and axonal repair by polyethylene glycol and methylene blue

Mammalian neurons and all other eukaryotic cells endogenously repair traumatic injury within minutes by a Ca²⁺-induced accumulation of vesicles that interact and fuse with each other and the plasmalemma to seal any openings. We have used uptake or exclusion of extracellular fluorescent dye to measure the ability of rat hippocampal B104 cells or rat sciatic nerves to repair (seal) transected neurites in vitro or transected axons ex vivo. We report that endogenous sealing in both preparations is enhanced by Ca²⁺-containing solutions and is decreased by Ca²⁺-free solutions containing antioxidants such as dithiothreitol (DTT), melatonin (MEL), methylene blue (MB), and various toxins that decrease vesicular interactions. In contrast, the fusogen polyethylene glycol (PEG) at 10-50 mM artificially seals the cut ends of B104 cells and rat sciatic axons within seconds and is not affected by Ca²⁺ or any of the substances that affect endogenous sealing. At higher concentrations, PEG decreases sealing of transected axons and disrupts the plasmalemma of intact cells. These PEG-sealing data are consistent with the hypothesis that lower concentrations of PEG directly seal a damaged plasmalemma. We have considered these and other data to devise a protocol using a well-specified series of solutions that vary in tonicity, Ca²⁺, MB, and PEG content. These protocols rapidly and consistently repair (PEG-fuse) rat sciatic axons in completely cut sciatic nerves in vivo rapidly and dramatically to restore long-lasting morphological continuity, action potential conduction, and behavioral functions.

Laser-based single-axon transection for high-content axon injury and regeneration studies.

The investigation of the regenerative response of the neurons to axonal injury is essential to the development of new axoprotective therapies. Here we study the retinal neuronal RGC-5 cell line after laser transection, demonstrating that the ability of these cells to initiate a regenerative response correlates with axon length and cell motility after injury. We show that low energy picosecond laser pulses can achieve transection of unlabeled single axons in vitro and precisely induce damage with micron precision. We established the conditions to achieve axon transection, and characterized RGC-5 axon regeneration and cell body response using time-lapse microscopy. We developed an algorithm to analyze cell trajectories and established correlations between cell motility after injury, axon length, and the initiation of the regeneration response. The characterization of the motile response of axotomized RGC-5 cells showed that cells that were capable of repair or regrowth of damaged axons migrated more slowly than cells that could not. Moreover, we established that RGC-5 cells with long axons could not recover their injured axons, and such cells were much more motile. The platform we describe allows highly controlled axonal damage with subcellular resolution and the performance of high-content screening in cell cultures.

Erythropoietin Enhances Nerve Repair in Anti-Ganglioside Antibody-Mediated Models of Immune Neuropathy

Guillain-Barré syndrome (GBS) is a monophasic immune neuropathic disorder in which a significant proportion of patients have incomplete recovery. The patients with incomplete recovery almost always have some degree of failure of axon regeneration and target reinnervation. Anti-ganglioside antibodies (Abs) are the most commonly recognized autoimmune markers in all forms of GBS and specific Abs are associated with the slow/poor recovery. We recently demonstrated that specific anti-ganglioside Abs inhibit axonal regeneration and nerve repair in preclinical models by activation of small GTPase RhoA and its downstream effectors. The objective of this study was to determine whether erythropoietin (EPO), a pleiotropic cytokine with neuroprotective and neurotrophic properties, enhances nerve regeneration in preclinical cell culture and animal models of autoimmune neuropathy/nerve repair generated with monoclonal and patient derived Abs. Primary neuronal cultures and a standardized sciatic crush nerve model were used to assess the efficacy of EPO in reversing inhibitory effects of anti-ganglioside Abs on nerve repair. We found that EPO completely reversed the inhibitory effects of anti-ganglioside Abs on axon regeneration in cell culture models and significantly improved nerve regeneration/repair in an animal model. Moreover, EPO-induced proregenerative effects in nerve cells are through EPO receptors and Janus kinase 2/Signal transducer and activator of transcription 5 pathway and not via early direct modulation of small GTPase RhoA. These preclinical studies indicate that EPO is a viable candidate drug to develop further for neuroprotection and enhancing nerve repair in patients with GBS.

GSK-3β activation mediates Nogo-66-induced inhibition of neurite outgrowth in N2a cells

The axons of the adult mammalian brain and spinal cord fail to regenerate after injury, and it has been suggested that Nogo-66 could prevent CNS axon repair. However, the mechanism of Nogo-66 inhibiting neurite outgrowth remains unknown. Our previous results indicated that protein kinase B (PKB) is involved in the inhibition of the neurite outgrowth by Nogo-66. Glycogen synthase kinase-3β (GSK-3β) is implicated in many processes in the nervous system, including differentiation, specification, polarity, plasticity and axon growth. In addition, GSK-3β is one of the most important molecules downstream of PKB. In the present study, we report on the role of GSK-3β signaling on Nogo-66-treated mouse neuroblastoma N2a cells. Nogo-66 reduced the phosphorylation of GSK-3β at Ser9 in N2a cells. In contrast, pretreatment with SB216763, a specific inhibitor of GSK-3β, resulted in an amelioration of neurite outgrowth by Nogo-66, compared with the Nogo-66 alone group (P<0.05). Moreover, we performed RNA interference experiments to knock down GSK-3β expression levels in N2a cells via transient transfection of shRNA plasmids. The inhibition of neurite outgrowth by Nogo-66 was subdued in shRNA cells, compared to the non-RNAi cells (P<0.05). Taken together, these data suggest that GSK-3β is involved in the inhibition by Nogo-66 of neurite outgrowth in N2a cells.

Neurite outgrowth is differentially impacted by distinct immune cell subsets

Axonal damage can occur in the central nervous system following trauma, during the course of autoimmune and neurodegenerative disease and during viral and bacterial infections. The degree of axonal damage and absence of spontaneous repair are major determinants of long-term clinical outcome. While inflammation is a common feature of these conditions, the impact of particular immune cell subsets and their products on injured axons is not fully known. To investigate the impact of immune cells on neuronal viability and axonal repair, we developed an in vitro culture system in which neurons are exposed to mixed or distinct immune cell subsets. We find that total peripheral blood mononuclear cells (PBMCs) have a significant inhibitory effect on neurite outgrowth that is independent of apoptosis. Using isolated immune cells subsets, we demonstrate that activated CD4+ T cells enhance neurite outgrowth while activated NK cells and CD8+ T cells inhibit neurite outgrowth. We find that NK cell inhibition of neuronal outgrowth is dependent on MAPK activity. Our findings describe heterogeneous effects of individual immune cell subsets on neuronal growth and offer important insights into the cellular and molecular mechanisms that may impact axonal repair in inflammatory CNS conditions.

FOXP3 gene expression in multiple sclerosis patients pre- and post mesenchymal stem cell therapy.

Multiple Sclerosis (MS) is an inflammatory demyelinating and neurodegenerative disorder of the central nervous system (CNS), which mainly affects young adults. Activated T lymphocytes promote the neuro-inflammatory cascade of MS by secreting pro-inflammatory cytokines and play a significant role in its pathogenesis. T lymphocytes may trigger the inflammation, which in turn leads to axonal loss and neurodegeneration observed in the course of MS. Currently, there is no cure for MS, however, one of the most promising neuroprotective research tools consists of the use of bone marrow derived mesenchymal stem cells (MSC). This method promotes immune system regulation and possibly induces neurological repair and re-myelination of the damaged axons. Recent studies have shown that MSC exert an immune regulatory function and induce T regulatory-cell proliferation, therefore, it may serve as a potentially useful treatment for immune-mediated diseases such as MS. In this pilot study a group of MS patients underwent MSC therapy and we assayed the expression of an X-linked transcription factor, FoxP3, as a specific marker of T Regulatory cells in peripheral blood, prior to and after the treatment. Using q RT-PCR for measurement of expression of FoxP3 by peripheral blood mononuclear cells, we found that in all subjects, except for one, the expression of FoxP3 at 6 months after intrathecal injection of MSC was significantly higher than the levels prior to treatment. Such significant enhanced expression of FoxP3 associated with clinical stability. Findings from this pilot study further support the potential of bone marrow derived MSC for treatment of MS patients.

Axon Regeneration Pathways Identified by Systematic Genetic Screening in C. elegans

The mechanisms underlying the ability of axons to regrow after injury remain poorly explored at the molecular genetic level. We used a laser injury model in Caenorhabditis elegans mechanosensory neurons to screen 654 conserved genes for regulators of axonal regrowth. We uncover several functional clusters of genes that promote or repress regrowth, including genes classically known to affect axon guidance, membrane excitability, neurotransmission, and synaptic vesicle endocytosis. The conserved Arf Guanine nucleotide Exchange Factor (GEF), EFA-6, acts as an intrinsic inhibitor of regrowth. By combining genetics and invivo imaging, we show that EFA-6 inhibits regrowth via microtubule dynamics, independent of its Arf GEF activity. Among newly identified regrowth inhibitors, only loss of function in EFA-6 partially bypasses the requirement for DLK-1 kinase. Identification of these pathways significantly expands our understanding of the genetic basis of axonal injury responses and repair.

Exposure Levels of Anti-LINGO-1 Li81 Antibody in the Central Nervous System and Dose-Efficacy Relationships in Rat Spinal Cord Remyelination Models after Systemic Administration

LINGO-1 (leucine-rich repeat and Ig domain containing NOGO receptor interacting protein-1) is a negative regulator of myelination and repair of damaged axons in the central nervous system (CNS). Blocking LINGO-1 function leads to robust remyelination. The anti-LINGO-1 Li81 antibody is currently being evaluated in clinical trials for multiple sclerosis (MS) and is the first MS therapy that directly targets myelin repair. LINGO-1 is selectively expressed in brain and spinal cord but not in peripheral tissues. Perhaps the greatest concern for Li81 therapy is the limited access of the drug to the CNS. Here, we measured Li81 concentrations in brain, spinal cord, and cerebral spinal fluid in rats after systemic administration and correlated them with dose-efficacy responses in rat lysolecithin and experimental autoimmune encephalomyelitis spinal cord models of remyelination. Remyelination was dose-dependent, and levels of Li81 in spinal cord that promoted myelination correlated well with affinity measurements for the binding of Li81 to LINGO-1. Observed Li81 concentrations in the CNS of 0.1 to 0.4% of blood levels are consistent with values reported for other antibodies. To understand the features of the antibody that affect CNS penetration, we also evaluated the pharmacokinetics of Li81 Fab2, Fab, and poly(ethylene glycol)-modified Fab. The reagents all showed similar CNS exposure despite large differences in their sizes, serum half-lives, and volumes of distribution, and area under the curve (AUC) measurements in the CNS directly correlated with AUC measurements in serum. These studies demonstrate that exposure levels achieved by passive diffusion of the Li81 monoclonal antibody into the CNS are sufficient and lead to robust remyelination.

Myeloid lineage cells inhibit neurite outgrowth through a myosin II-dependent mechanism

The molecular mechanisms that underlie the axonal damage that accompanies CNS inflammation are largely unknown. Here, we investigate the effects of immune cells on neuronal viability and axonal growth and show that conditioned media from myeloid lineage cells inhibit neurite outgrowth without causing apoptosis. Treatment with monocyte conditioned medium enhances myosin light chain phosphorylation in neurons and the neurite outgrowth inhibitory effect of myeloid lineage cells can be attenuated with the myosin II inhibitor blebbistatin. Our results suggest that in the context of CNS inflammation myeloid cells may limit axonal repair in the CNS via a myosin II-dependent mechanism.

Chondroitinase activity can be transduced by a lentiviral vector in vitro and in vivo

The bacterial enzyme chondroitinase ABC (ChABC), which cleaves chondroitin sulfate glycosaminoglycan chains, can degrade inhibitory scar tissue formed following spinal cord injury, thereby promoting axonal growth and regeneration. However, delivering the active enzyme for prolonged periods presents practical limitations. To overcome these problems, we prepared a lentiviral vector (LV) encoding chondroitinase AC (Chase) together with the green fluorescent protein (GFP) reporter (Chase/LV) and demonstrated its expression and enzymatic activity in vitro and in vivo. Neural precursor cells infected with Chase/LV expressed the GFP reporter at levels that increased dramatically with time in culture. Enzymatic activity from the supernatant of the infected cells was demonstrated by dot blot assay using an antibody that recognizes the digested form of CSPG and was compared with the bacterial ChABC enzyme. Chick DRG cultures plated adjacent to the CSPG border and incubated with supernatant from Chase/LV-infected cells showed neurites growing into the CSPG area, a response similar to that after treatment with ChABC. In contrast, in control cultures, the neurites turned to avoid the inhibitory CSPG interface. Degradation of CSPG in these cultures was confirmed by specific CSPG antibodies. A single injection of Chase/LV into the spinal cord resulted in sustained secretion of the enzyme, whose activity was detected for 8 weeks by expression of GFP and evidence of the digested form of CSPG. This study demonstrates the efficacy of the Chase/LV vector and its potential as a therapeutic tool to reduce scar inhibition and promote axonal growth and repair following central nervous system injury.

Human neuropathological and animal model evidence supporting a role for Fas-mediated apoptosis and inflammation in cervical spondylotic myelopathy

Although cervical spondylotic myelopathy is a common cause of chronic spinal cord dysfunction in humans, little is known about the molecular mechanisms underlying the progressive neural degeneration characterized by this condition. Based on animal models of cervical spondylotic myelopathy and traumatic spinal cord injury, we hypothesized that Fas-mediated apoptosis and inflammation may play an important role in the pathobiology of human cervical spondylotic myelopathy. We further hypothesized that neutralization of the Fas ligand using a function-blocking antibody would reduce cell death, attenuate inflammation, promote axonal repair and enhance functional neurological outcomes in animal models of cervical spondylotic myelopathy. We examined molecular changes in post-mortem human spinal cord tissue from eight patients with cervical spondylotic myelopathy and four control cases. Complementary studies were conducted using a mouse model of cervical spondylotic myelopathy (twy/twy mice that develop spontaneous cord compression at C2-C3). We observed Fas-mediated apoptosis of neurons and oligodendrocytes and an increase in inflammatory cells in the compressed spinal cords of patients with cervical spondylotic myelopathy. Furthermore, neutralization of Fas ligand with a function-blocking antibody in twy/twy mice reduced neural inflammation at the lesion mediated by macrophages and activated microglia, glial scar formation and caspase-9 activation. It was also associated with increased expression of Bcl-2 and promoted dramatic functional neurological recovery. Our data demonstrate, for the first time in humans, the potential contribution of Fas-mediated cell death and inflammation to the pathobiology of cervical spondylotic myelopathy. Complementary data in a murine model of cervical spondylotic myelopathy further suggest that targeting the Fas death receptor pathway is a viable neuroprotective strategy to attenuate neural degeneration and optimize neurological recovery in cervical spondylotic myelopathy. Our findings highlight the possibility of medical treatments for cervical spondylotic myelopathy that are complementary to surgical decompression.

Single-Walled Carbon Nanotubes Chemically Functionalized with Polyethylene Glycol Promote Tissue Repair in a Rat Model of Spinal Cord Injury

Traumatic spinal cord injury (SCI) induces tissue damage and results in the formation of a cavity that inhibits axonal regrowth. Filling this cavity with a growth-permissive substrate would likely promote regeneration and repair. Single-walled carbon nanotubes functionalized with polyethylene glycol (SWNT-PEG) have been shown to increase the length of selected neurites in vitro. We hypothesized that administration of SWNT-PEG after experimental SCI will promote regeneration of axons into the lesion cavity and functional recovery of the hindlimbs. To evaluate this hypothesis, complete transection SCI was induced at the T9 vertebral level in adult female rats. One week after transection, the epicenter of the lesion was injected with 25??L of either vehicle (saline), or 1??g/mL, 10??g/mL, or 100??g/mL of SWNT-PEG. Behavioral analysis was conducted before injury, before treatment, and once every 7 days for 28 days after treatment. At 28 days post-injection the rats were euthanized and spinal cord tissue was extracted. Immunohistochemistry was used to detect the area of the cyst, the extent of the glial scar, and axonal morphology. We found that post-SCI administration of SWNT-PEG decreased lesion volume, increased neurofilament-positive fibers and corticospinal tract fibers in the lesion, and did not increase reactive gliosis. Additionally, post-SCI administration of SWNT-PEG induced a modest improvement in hindlimb locomotor recovery without inducing hyperalgesia. These data suggest that SWNT-PEG may be an effective material to promote axonal repair and regeneration after SCI.

Diffuse Traumatic Axonal Injury in the Mouse Induces Atrophy, c-Jun Activation, and Axonal Outgrowth in the Axotomized Neuronal Population

Traumatic axonal injury (TAI) is a consistent component of traumatic brain injury (TBI) and is associated with much of its morbidity. Little is known regarding the long-term retrograde neuronal consequences of TAI and/or the potential that TAI could lead to anterograde axonal reorganization and repair. To investigate the repertoire of anterograde and retrograde responses triggered by TIA, Thy1-YFP-H mice were subjected to mild central fluid percussion injury and killed at various times between 15 min and 28 d post-injury. Based upon confocal assessment of the endogenous neuronal fluorescence, such injury was found to result in diffuse TAI throughout layer V of the neocortex within yellow fluorescent protein (YFP)-positive axons. When these fluorescent approaches were coupled with various quantitative and immunohistochemical approaches, we found that this TAI did not result in neuronal death over the 28 d period assessed. Rather, it elicited neuronal atrophy. Within these same axotomized neuronal populations, TAI was also found to induce an early and sustained activation of the transcription factors c-Jun and ATF-3 (activating transcription factor 3), known regulators of axon regeneration. Parallel ultrastructural studies confirmed that these reactive changes are consistent with atrophy in the absence of neuronal death. Concurrent with those events ongoing in the neuronal cell bodies, their downstream axonal segments revealed, as early as 1 d post-injury, morphological changes consistent with reactive sprouting that was accompanied by significant axonal elongation over time. Collectively, these TAI-linked events are consistent with sustained neuronal recovery, an activation of a regenerative genetic program, and subsequent axonal reorganization suggestive of some form of regenerative response.

Axotomy-induced cytoskeleton changes in unmyelinated mammalian central nervous system axons

Oligodendrocyte-derived myelin retards the ability of CNS axons to regenerate following transection. The intrinsic response of CNS axons to an axotomy insult may be vastly different in the absence of myelin. However, the paucity of adequate experimental models has limited detailed investigation of cellular behaviour following axon transection in an unmyelinated CNS environment. In this study we perform laser-induced axotomy of the porcine retinal ganglion cell axon, a physiologically unmyelinated, mature CNS axon that is structurally similar to humans to infer knowledge about axonal behaviour in the absence of myelin. Axotomy-induced changes to the neuronal cytoskeleton and supporting astrocytes during the early stages after transection are delineated by examining the sequence of neurofilament subunit, microtubule (TUB), microtubule associated protein (MAP), glial fibrillary acidic protein (GFAP) and terminal deoxynucleotidyl transferase biotin-dUTP nick end labelling (TUNEL) modification. Axonal transection induced an increase in the expression of neurofilament light at regions within, and immediately adjacent to, sites of axotomy. Other neurofilament subunits were not altered at sites of transection. Unlike myelinated axons where an increase in GFAP staining within hypertrophic glial scars have been shown to inhibit axonal repair we demonstrate a decrease in GFAP staining within regions of increased or preserved neurofilament expression. The behaviour of TUB and MAP proteins following transection of unmyelinated CNS axons are similar to what has previously been described in myelinated CNS axons. This study provides fundamental insights into astrocyte and axonal behaviour acutely after axotomy and demonstrates a series of degenerative events in unmyelinated CNS axons, which in comparison to prior reports are different to myelinated CNS axons. The findings of this report have relevance to understanding pathogenic mechanisms underlying neuro-degeneration in the CNS.