The role of RhoA in retrograde neuronal death and axon regeneration after spinal cord injury.

Paralysis following spinal cord injury (SCI) is due to interruption of axons and their failure to regenerate. It has been suggested that the small GTPase RhoA may be an intracellular signaling convergence point for several types of growth-inhibiting extracellular molecules. Even if this is true in vitro, it is not clear from studies in mammalian SCI, whether the effects of RhoA manipulations on axon growth in vivo are due to a RhoA-mediated inhibition of true regeneration or only of collateral sprouting from spared axons, since work on SCI generally is performed with partial injury models. RhoA also has been implicated in local neuronal apoptosis after SCI, but whether this reflects an effect on axotomy-induced cell death or an effect on other pathological mechanisms is not known. In order to resolve these ambiguities, we studied the effects of RhoA knockdown in the sea lamprey central nervous system (CNS), where after complete spinal cord transection (TX), robust but incomplete regeneration of large axons belonging to individually identified reticulospinal (RS) neurons occurs, and where some RS neurons show unambiguous delayed retrograde apoptosis after axotomy. RhoA protein was detected in neurons and axons of the lamprey brain and spinal cord, and its expression was increased post-TX. Knockdown of RhoA in vivo by retrogradely-delivered morpholino antisense oligonucleotides (MOs) to the RS neurons significantly reduced retrograde apoptosis signaling in identified RS neurons post-SCI, as indicated by Fluorochrome Labeled Inhibitor of Caspases (FLICA) in brain wholemounts. In individual RS neurons, the reduction of caspase activation by RhoA knockdown began at 2weeks post-TX and was still seen at 8weeks. RhoA knockdown slowed axon retraction and possibly increased early axon regeneration in the proximal stump. The number of axons regenerating beyond the lesion more than 5mm at 10weeks post-TX also was increased. Thus RhoA knockdown both enhanced true axon regeneration and inhibited retrograde apoptosis signaling after SCI.

Increased mortality persists in an adult drug-resistant epilepsy prevalence cohort.

OBJECTIVE: To investigate the cumulative probability of death and the standardised mortality ratio (SMR) in an adult drug-resistant epilepsy (DRE) population. METHODS: In two separate centres during 2003-2006, we identified a total of 433 patients with DRE defined as at least one seizure per month and failure of at least two antiepileptic drugs. These patients were subsequently followed for a total follow-up of 6 years. We examined the cumulative probability of death, using Kaplan-Meier methodology, and the SMR based on mortality data from the Social Security Death Index. Clinical predictors of death were evaluated using Cox regression analysis. RESULTS: The cumulative probability of death was 8.7% (95% CI 6.2% to 12.1%) at 6 years. The overall SMR was 2.4 (95% CI 1.7 to 3.3). It was 3.1; 95% CI 2.0 to 4.6 in subjects with remote or progressive aetiology and 1.7; 95% CI 0.8 to 2.8 in subjects with unknown aetiology. The SMR was significantly increased in those with a known remote aetiology (2.5; 95% CI (1.4 to 3.8)). Older age at enrolment and symptomatic generalised epilepsy syndrome were significant predictors of death. DISCUSSION: Mortality is increased in this drug-resistant population; largely driven by those with a known epilepsy aetiology. The increased mortality remains even after exclusion of those with a progressive aetiology. Previous studies of incident epilepsy cohorts revealed increased mortality that declines to near-normal levels after the first several years, but in our DRE cohort, mortality remains elevated despite a median duration of epilepsy of 25 years at study entry.

Combined RhoA morpholino and ChABC treatment protects identified lamprey neurons from retrograde apoptosis after spinal cord injury.

Previously, we reported that RhoA knockdown by morpholino antisense oligonucleotides (MOs), and enzymatic digestion of chondroitin sulfate proteoglycans (CSPGs) at the site of injury with chondroitinase ABC (ChABC), each can reduce retrograde neuronal apoptosis after spinal cord transection in the lamprey. To elucidate the mechanisms in neuronal survival and axon regeneration, we have investigated whether these two effects are additive in vivo. We used lampreys as a spinal cord injury model. MOs were used to knockdown RhoA and Chondroitinase ABC (ChABC) was used to digest CSPGs in vivo. Retrograde labeling, fluorochrome-labeled inhibitor of caspase activity (FLICA), immunohistochemistry, and western blots were performed to assess axonal regeneration, neuronal apoptotic signaling and Akt activation. Four treatment combinations were evaluated at 2-, 4-, and 10-weeks post-transection: (1) Control MO plus enzyme buffer (Ctrl); (2) control MO plus ChABC; (3) RhoA MO plus enzyme buffer (RhoA MO); and (4) RhoA MO plus ChABC (RhoA MO + ChABC). Consistent with our previous findings, at 4-weeks post-transection, there was less caspase activation in the ChABC and RhoA MO groups than in the Ctrl group. Moreover, the RhoA MO plus ChABC group had the best protective effect on identified reticulospinal (RS) neurons among the four treatment combinations. At 2 weeks post-transection, when axons have retracted maximally in the rostral stump and are beginning to regenerate back toward the lesion, the axon tips in the three treatment groups each were closer to the transection than those in the Ctr MO plus enzyme buffer group. Long-term axon regeneration also was evaluated for the large, individually identified RS neurons at 10 weeks post-transection by retrograde labeling. The percent regenerated axons in the RhoA MO plus ChABC group was greater than that in any of the other groups. Akt phosphorylation levels at threonine 308 was quantified in the identified RS neurons by western blots and immunofluorescence. The RhoA MO plus ChABC treatment enhanced pAkt-308 phosphorylation more than any of the other treatment groups. Although some of the effects of CSPGs are mediated through RhoA activation, some growth-inhibiting mechanisms of RhoA and CSPGs are independent of each other, so combinatorial therapies may be warranted.

The Composition and Cellular Sources of CSPGs in the Glial Scar After Spinal Cord Injury in the Lamprey.

Axon regrowth after spinal cord injury (SCI) is inhibited by several types of inhibitory extracellular molecules in the central nervous system (CNS), including chondroitin sulfate proteoglycans (CSPGs), which also are components of perineuronal nets (PNNs). The axons of lampreys regenerate following SCI, even though their spinal cords contain CSPGs, and their neurons are enwrapped by PNNs. Previously, we showed that by 2 weeks after spinal cord transection in the lamprey, expression of CSPGs increased in the lesion site, and thereafter, decreased to pre-injury levels by 10 weeks. Enzymatic digestion of CSPGs in the lesion site with chondroitinase ABC (ChABC) enhanced axonal regeneration after SCI and reduced retrograde neuronal death. Lecticans (aggrecan, versican, neurocan, and brevican) are the major CSPG family in the CNS. Previously, we cloned a cDNA fragment that lies in the most conserved link-domain of the lamprey lecticans and found that lectican mRNAs are expressed widely in lamprey glia and neurons. Because of the lack of strict one-to-one orthology with the jawed vertebrate lecticans, the four lamprey lecticans were named simply A, B, C, and D. Using probes that distinguish these four lecticans, we now show that they all are expressed in glia and neurons but at different levels. Expression levels are relatively high in embryonic and early larval stages, gradually decrease, and are upregulated again in adults. Reductions of lecticans B and D are greater than those of A and C. Levels of mRNAs for lecticans B and D increased dramatically after SCI. Lectican D remained upregulated for at least 10 weeks. Multiple cells, including glia, neurons, ependymal cells and microglia/macrophages, expressed lectican mRNAs in the peripheral zone and lesion center after SCI. Thus, as in mammals, lamprey lecticans may be involved in axon guidance and neuroplasticity early in development. Moreover, neurons, glia, ependymal cells, and microglia/macrophages, are responsible for the increase in CSPGs during the formation of the glial scar after SCI.

Remission and relapse in a drug-resistant epilepsy population followed prospectively.

PURPOSE: We investigated the cumulative probability of seizure remission and relapse in an adult population with drug-resistant epilepsy and frequent seizures. In addition, we determined clinical predictors of remission and relapse in this population. METHODS: IN 2003, we identified 246 patients at a single center with drug-resistant epilepsy defined as at least one seizure per month and failure of at least two antiepileptic drugs. These patients were followed prospectively (cohort design). We examined the cumulative probability of seizure remission and relapse in this population using Kaplan-Meier methodology. Clinical predictors of remission and relapse were also evaluated using Cox regression analysis. KEY FINDINGS: The estimated cumulative probability of 12-month seizure remission was 34.6% at 7 years in the entire population and 33.4% when limited to those without surgery. The risk for relapse after a 12-month period of seizure remission was 71.2% at 5 years. Negative predictors of seizure remission included developmental delay, symptomatic generalized epilepsy syndrome, duration of intractability, and number of antiepileptic drugs failed. Localization-related epilepsy was the only negative predictor of relapse. SIGNIFICANCE: Among patients with drug-resistant epilepsy, 5% per year enter seizure remission even with a follow-up of 6 years. However, a substantial proportion of these patients relapse after the first year following a remission. The large proportion of patients entering a significant remission gives these patients hope; however, caution should be advised when discussing the likelihood of future seizures.

Chondroitinase ABC Promotes Axon Regeneration and Reduces Retrograde Apoptosis Signaling in Lamprey.

Paralysis following spinal cord injury (SCI) is due to failure of axonal regeneration. It is believed that axon growth is inhibited by the presence of several types of inhibitory molecules in central nervous system (CNS), including the chondroitin sulfate proteoglycans (CSPGs). Many studies have shown that digestion of CSPGs with chondroitinase ABC (ChABC) can enhance axon growth and functional recovery after SCI. However, due to the complexity of the mammalian CNS, it is still unclear whether this involves true regeneration or only collateral sprouting by uninjured axons, whether it affects the expression of CSPG receptors such as protein tyrosine phosphatase sigma (PTPσ), and whether it influences retrograde neuronal apoptosis after SCI. In the present study, we assessed the roles of CSPGs in the regeneration of spinal-projecting axons from brainstem neurons, and in the process of retrograde neuronal apoptosis. Using the fluorochrome-labeled inhibitor of caspase activity (FLICA) method, apoptotic signaling was seen primarily in those large, individually identified reticulospinal (RS) neurons that are known to be "bad-regenerators." Compared to uninjured controls, the number of all RS neurons showing polycaspase activity increased significantly at 2, 4, 8, and 11 weeks post-transection (post-TX). ChABC application to a fresh TX site reduced the number of polycaspase-positive RS neurons at 2 and 11 weeks post-TX, and also reduced the number of active caspase 3-positive RS neurons at 4 weeks post-TX, which confirmed the beneficial role of ChABC treatment in retrograde apoptotic signaling. ChABC treatment also greatly promoted axonal regeneration at 10 weeks post-TX. Correspondingly, PTPσ mRNA expression was reduced in the perikaryon. Previously, PTPσ mRNA expression was shown to correlate with neuronal apoptotic signaling at 2 and 10 weeks post-TX. In the present study, this correlation persisted after ChABC treatment, which suggests that PTPσ may be involved more generally in signaling axotomy-induced retrograde neuronal apoptosis. Moreover, ChABC treatment caused Akt activation (pAkt-308) to be greatly enhanced in brain post-TX, which was further confirmed in individually identified RS neurons. Thus, CSPG digestion not only enhances axon regeneration after SCI, but also inhibits retrograde RS neuronal apoptosis signaling, possibly by reducing PTPσ expression and enhancing Akt activation.

Mechanisms of Axon Elongation Following CNS Injury: What Is Happening at the Axon Tip?

After an injury to the central nervous system (CNS), functional recovery is limited by the inability of severed axons to regenerate and form functional connections with appropriate target neurons beyond the injury. Despite tremendous advances in our understanding of the mechanisms of axon growth, and of the inhibitory factors in the injured CNS that prevent it, disappointingly little progress has been made in restoring function to human patients with CNS injuries, such as spinal cord injury (SCI), through regenerative therapies. Clearly, the large number of overlapping neuron-intrinsic and -extrinsic growth-inhibitory factors attenuates the benefit of neutralizing any one target. More daunting is the distances human axons would have to regenerate to reach some threshold number of target neurons, e.g., those that occupy one complete spinal segment, compared to the distances required in most experimental models, such as mice and rats. However, the difficulties inherent in studying mechanisms of axon regeneration in the mature CNS in vivo have caused researchers to rely heavily on extrapolation from studies of axon regeneration in peripheral nerve, or of growth cone-mediated axon development in vitro and in vivo. Unfortunately, evidence from several animal models, including the transected lamprey spinal cord, has suggested important differences between regeneration of mature CNS axons and growth of axons in peripheral nerve, or during embryonic development. Specifically, long-distance regeneration of severed axons may not involve the actin-myosin molecular motors that guide embryonic growth cones in developing axons. Rather, non-growth cone-mediated axon elongation may be required to propel injured axons in the mature CNS. If so, it may be necessary to use other experimental models to promote regeneration that is sufficient to contact a critical number of target neurons distal to a CNS lesion. This review examines the cytoskeletal underpinnings of axon growth, focusing on the elongating axon tip, to gain insights into how CNS axons respond to injury, and how this might affect the development of regenerative therapies for SCI and other CNS injuries.

Heterogeneity in the regenerative abilities of central nervous system axons within species: why do some neurons regenerate better than others?

Some neurons, especially in mammalian peripheral nervous system or in lower vertebrate or in vertebrate central nervous system (CNS) regenerate after axotomy, while most mammalian CNS neurons fail to regenerate. There is an emerging consensus that neurons have different intrinsic regenerative capabilities, which theoretically could be manipulated therapeutically to improve regeneration. Population-based comparisons between "good regenerating" and "bad regenerating" neurons in the CNS and peripheral nervous system of most vertebrates yield results that are inconclusive or difficult to interpret. At least in part, this reflects the great diversity of cells in the mammalian CNS. Using mammalian nervous system imposes several methodical limitations. First, the small sizes and large numbers of neurons in the CNS make it very difficult to distinguish regenerating neurons from non-regenerating ones. Second, the lack of identifiable neurons makes it impossible to correlate biochemical changes in a neuron with axonal damage of the same neuron, and therefore, to dissect the molecular mechanisms of regeneration on the level of single neurons. This review will survey the reported responses to axon injury and the determinants of axon regeneration, emphasizing non-mammalian model organisms, which are often under-utilized, but in which the data are especially easy to interpret.

Source of Early Regenerating Axons in Lamprey Spinal Cord Revealed by Wholemount Optical Clearing with BABB.

Many studies of axon regeneration in the lamprey focus on 18 pairs of large identified reticulospinal (RS) neurons, whose regenerative abilities have been individually quantified. Their axons retract during the first 2 weeks after transection (TX), and many grow back to the site of injury by 4 weeks. However, locomotor movements begin before 4 weeks and the lesion is invaded by axons as early as 2 weeks post-TX. The origins of these early regenerating axons are unknown. Their identification could be facilitated by studies in central nervous system (CNS) wholemounts, particularly if spatial resolution and examination by confocal microscopy were not limited by light scattering. We have used benzyl alcohol/benzyl benzoate (BABB) clearing to enhance the resolution of neuronal perikarya and regenerated axons by confocal microscopy in lamprey CNS wholemounts, and to assess axon regeneration by retrograde and anterograde labeling with fluorescent dye applied to a second TX caudal or rostral to the original lesion, respectively. We found that over 50% of the early regenerating axons belonged to small neurons in the brainstem. Some propriospinal neurons located close to the TX also contributed to early regeneration. The number of early regenerating propriospinal neurons decreased with distance from the original lesion. Descending axons from the brainstem were labeled anterogradely by application of tracer to a second TX close to the spinal-medullary junction. This limited contamination of the data by regenerating spinal axons whose cell bodies are located rostral or caudal to the TX and confirmed the regeneration of many small RS axons as early as 2 weeks post-TX. Compared with the behavior of axotomized giant axons, the early regenerating axons were of small caliber and showed little retraction, probably because they resealed rapidly after injury.

PTPσ Knockdown in Lampreys Impairs Reticulospinal Axon Regeneration and Neuronal Survival After Spinal Cord Injury.

Traumatic spinal cord injury (SCI) results in persistent functional deficits due to the lack of axon regeneration within the mammalian CNS. After SCI, chondroitin sulfate proteoglycans (CSPGs) inhibit axon regrowth via putative interactions with the LAR-family protein tyrosine phosphatases, PTPσ and LAR, localized on the injured axon tips. Unlike mammals, the sea lamprey, Petromyzon marinus, robustly recovers locomotion after complete spinal cord transection (TX). Behavioral recovery is accompanied by heterogeneous yet predictable anatomical regeneration of the lamprey's reticulospinal (RS) system. The identified RS neurons can be categorized as "good" or "bad" regenerators based on the likelihood that their axons will regenerate. Those neurons that fail to regenerate their axons undergo a delayed form of caspase-mediated cell death. Previously, this lab reported that lamprey PTPσ mRNA is selectively expressed in "bad regenerator" RS neurons, preceding SCI-induced caspase activation. Consequently, we hypothesized that PTPσ deletion would reduce retrograde cell death and promote axon regeneration. Using antisense morpholino oligomers (MOs), we knocked down PTPσ expression after TX and assessed the effects on axon regeneration, caspase activation, intracellular signaling, and behavioral recovery. Unexpectedly, PTPσ knockdown significantly impaired RS axon regeneration at 10 weeks post-TX, primarily due to reduced long-term neuron survival. Interestingly, cell loss was not preceded by an increase in caspase or p53 activation. Behavioral recovery was largely unaffected, although PTPσ knockdowns showed mild deficits in the recovery of swimming distance and latency to immobility during open field swim assays. Although the mechanism underlying the cell death following TX and PTPσ knockdown remains unknown, this study suggests that PTPσ is not a net negative regulator of long tract axon regeneration in lampreys.

Role of axon resealing in retrograde neuronal death and regeneration after spinal cord injury.

Spinal cord injury leads to persistent behavioral deficits because mammalian central nervous system axons fail to regenerate. A neuron's response to axon injury results from a complex interplay of neuron-intrinsic and environmental factors. The contribution of axotomy to the death of neurons in spinal cord injury is controversial because very remote axotomy is unlikely to result in neuronal death, whereas death of neurons near an injury may reflect environmental factors such as ischemia and inflammation. In lampreys, axotomy due to spinal cord injury results in delayed apoptosis of spinal-projecting neurons in the brain, beyond the extent of these environmental factors. This retrograde apoptosis correlates with delayed resealing of the axon, and can be reversed by inducing rapid membrane resealing with polyethylene glycol. Studies in mammals also suggest that polyethylene glycol may be neuroprotective, although the mechanism(s) remain unclear. This review examines the early, mechanical, responses to axon injury in both mammals and lampreys, and the potential of polyethylene glycol to reduce injury-induced pathology. Identifying the mechanisms underlying a neuron's response to axotomy will potentially reveal new therapeutic targets to enhance regeneration and functional recovery in humans with spinal cord injury.

The Effect of Axon Resealing on Retrograde Neuronal Death after Spinal Cord Injury in Lamprey.

Failure of axon regeneration in the central nervous system (CNS) of mammals is due to both extrinsic inhibitory factors and to neuron-intrinsic factors. The importance of intrinsic factors is illustrated in the sea lamprey by the 18 pairs of large, individually identified reticulospinal (RS) neurons, whose axons are located in the same spinal cord tracts but vary greatly in their ability to regenerate after spinal cord transection (TX). The neurons that are bad regenerators also undergo very delayed apoptosis, signaled early by activation of caspases. We noticed that the neurons with a low probability of axon regeneration tend to be larger than the good regenerators. We postulate that the poorly regenerating larger neurons have larger caliber axons, which reseal more slowly, allowing more prolonged entry of toxic signals (e.g., Ca++) into the axon at the injury site. To test this hypothesis, we used a dye-exclusion assay, applying membrane-impermeable dyes to the cut ends of spinal cords at progressively longer post-TX intervals. Axons belonging to the very small neurons (not individually identified) of the medial inferior RS nucleus resealed within 15 min post-TX. Almost 75% of axons belonging to the medium-sized identified RS neurons resealed within 3 h. At this time, only 36% of the largest axons had resealed, often taking more than 24 h to exclude the dye. There was an inverse relationship between an RS neuron's size and the probability that its axon would regenerate (r = -0.92) and that the neuron would undergo delayed apoptosis, as indicated by staining with a fluorescently labeled inhibitor of caspases (FLICA; r = 0.73). The artificial acceleration of resealing with polyethylene glycol (PEG) reduced retrograde neuronal apoptosis by 69.5% at 2 weeks after spinal cord injury (SCI), suggesting that axon resealing is a critical determinant of cell survival. Ca++-free Ringer's solution with EGTA prolonged the sealing time and increased apoptotic signaling, suggesting that factors other than Ca++ diffusion into the injured tip contribute to retrograde death signaling. A longer distance of the lesion from the cell body reduced apoptotic signaling independent of the axon sealing time.

RhoA activation in axotomy-induced neuronal death.

After spinal cord injury (SCI) in mammals, severed axons fail to regenerate, due to both extrinsic inhibitory factors, e.g., the chondroitin sulfate proteoglycans (CSPGs) and myelin-associated growth inhibitors (MAIs), and a developmental loss of intrinsic growth capacity. The latter is suggested by findings in lamprey that the 18 pairs of individually identified reticulospinal neurons vary greatly in their ability to regenerate their axons through the same spinal cord environment. Moreover, those neurons that are poor regenerators undergo very delayed apoptosis, and express common molecular markers after SCI. Thus the signaling pathways for retrograde cell death might converge with those inhibiting axon regeneration. Many extrinsic growth-inhibitory molecules activate RhoA, whereas inhibiting RhoA enhances axon growth. Whether RhoA also is involved in retrograde neuronal death after axotomy is less clear. Therefore, we cloned lamprey RhoA and correlated its mRNA expression and activation state with apoptosis signaling in identified reticulospinal neurons. RhoA mRNA was expressed widely in normal lamprey brain, and only slightly more in poorly-regenerating neurons than in good regenerators. However, within a day after spinal cord transection, RhoA mRNA was found in severed axon tips. Beginning at 5 days post-SCI RhoA mRNA was upregulated selectively in pre-apoptotic neuronal perikarya, as indicated by labelling with fluorescently labeled inhibitors of caspase activation (FLICA). After 2 weeks post-transection, RhoA expression decreased in the perikarya, and was translocated anterogradely into the axons. More striking than changes in RhoA mRNA levels, RhoA was continuously active selectively in FLICA-positive neurons through 9 weeks post-SCI. At that time, almost no neurons whose axons had regenerated were FLICA-positive. These findings are consistent with a role for RhoA activation in triggering retrograde neuronal death after SCI, and suggest that RhoA may be a point of convergence for inhibition of both axon regeneration and neuronal survival after axotomy.

Protein synthetic machinery and mRNA in regenerating tips of spinal cord axons in lamprey.

Polyribosomes, mRNA, and other elements of translational machinery have been reported in peripheral nerves and in elongating injured axons of sensory neurons in vitro, primarily in growth cones. Evidence for involvement of local protein synthesis in regenerating central nervous system (CNS) axons is less extensive. We monitored regeneration of back-labeled lamprey spinal axons after spinal cord transection and detected mRNA in axon tips by in situ hybridization and microaspiration of their axoplasm. Poly(A)+mRNA was present in the axon tips, and was more abundant in actively regenerating tips than in static or retracting ones. Target-specific polymerase chain reaction (PCR) and in situ hybridization revealed plentiful mRNA for the low molecular neurofilament subunit and ß-tubulin, but very little for ß-actin, consistent with the morphology of their tips, which lack filopodia and lamellipodia. Electron microscopy showed ribosomes/polyribosomes in the distal parts of axon tips and in association with vesicle-like membranes, primarily in the tip. In one instance, there were structures with the appearance of rough endoplasmic reticulum. Immunohistochemistry showed patches of ribosomal protein S6 positivity in a similar distribution. The results suggest that local protein synthesis might be involved in the mechanism of axon regeneration in the lamprey spinal cord. J. Comp. Neurol. 524:3614-3640, 2016. © 2016 Wiley Periodicals, Inc.

Antisense Morpholino Oligonucleotides Reduce Neurofilament Synthesis and Inhibit Axon Regeneration in Lamprey Reticulospinal Neurons.

The sea lamprey has been used as a model for the study of axonal regeneration after spinal cord injury. Previous studies have suggested that, unlike developing axons in mammal, the tips of regenerating axons in lamprey spinal cord are simple in shape, packed with neurofilaments (NFs), and contain very little F-actin. Thus it has been proposed that regeneration of axons in the central nervous system of mature vertebrates is not based on the canonical actin-dependent pulling mechanism of growth cones, but involves an internal protrusive force, perhaps generated by the transport or assembly of NFs in the distal axon. In order to assess this hypothesis, expression of NFs was manipulated by antisense morpholino oligonucleotides (MO). A standard, company-supplied MO was used as control. Axon retraction and regeneration were assessed at 2, 4 and 9 weeks after MOs were applied to a spinal cord transection (TX) site. Antisense MO inhibited NF180 expression compared to control MO. The effect of inhibiting NF expression on axon retraction and regeneration was studied by measuring the distance of axon tips from the TX site at 2 and 4 weeks post-TX, and counting the number of reticulospinal neurons (RNs) retrogradely labeled by fluorescently-tagged dextran injected caudal to the injury at 9 weeks post-TX. There was no statistically significant effect of MO on axon retraction at 2 weeks post-TX. However, at both 4 and 9 weeks post-TX, inhibition of NF expression inhibited axon regeneration.