Transient presence and functional interaction of endogenous GABA and GABAA receptors in developing rat optic nerve.

GABA (gamma-aminobutyric acid) is a major inhibitory synaptic neurotransmitter with widespread distribution in the central nervous system (CNS). GABA can also modulate axonal excitability by activation of GABAA receptors in CNS white matter regions where synapses and neuronal cell bodies are not present. Studies on cultured glia cells have revealed the synthesis of GABA in rat optic nerve O-2A progenitor cells that give rise to oligodendrocytes and type 2 astrocytes in vitro. We report here that: (i) GABA is detected by immuno-electron microscopy in intact rat optic nerve and is localized to glia and pre-myelinated axons during the first few weeks of postnatal development, but is markedly reduced or absent in the adult; and (ii) neonatal optic nerve is depolarized by GABAA receptor agonists or by the inhibition of GABA uptake. These results demonstrate the presence of functional GABAA receptors, and GABA uptake and release mechanisms in developing rat optic nerve, and suggest that excitability of developing axons can be modulated by endogenous neurotransmitter at non-synaptic sites.     

Traumatology of the optic nerve and contribution of crystallins to axonal regeneration

Within a few decades, the repair of long neuronal pathways such as spinal cord tracts, the optic nerve or intracerebral tracts has gone from being strongly contested to being recognized as a potential clinical challenge. Cut axonal stumps within the optic nerve were originally thought to retract and become irreversibly necrotic within the injury zone. Optic nerve astrocytes were assumed to form a gliotic scar and remodelling of the extracellular matrix to result in a forbidden environment for re-growth of axons. Retrograde signals to the ganglion cell bodies were considered to prevent anabolism, thus also initiating apoptotic death and gliotic repair within the retina. However, increasing evidence suggests the reversibility of these regressive processes, as shown by the analysis of molecular events at the site of injury and within ganglion cells. We review optic nerve repair from the perspective of the proximal axon stump being a major player in determining the successful formation of a growth cone. The axonal stump and consequently the prospective growth cone, communicates with astrocytes, microglial cells and the extracellular matrix via a panoply of molecular tools. We initially highlight these aspects on the basis of recent data from numerous laboratories. Then, we examine the mechanisms by which an injury-induced growth cone can sense its surroundings within the area distal to the injury. Based on requirements for successful axonal elongation within the optic nerve, we explore the models employed to instigate successful growth cone formation by ganglion cell stimulation and optic nerve remodelling, which in turn accelerate growth. Ultimately, with regard to the proteomics of regenerating retinal tissue, we discuss the discovery of isoforms of crystallins, with crystallin beta-b2 (crybb2) being clearly upregulated in the regenerating retina. Crystallins are produced and used to promote the elongation of growth cones. In vivo and in vitro, crystallins beta and gamma additionally promote the growth of axons by enhancing the production of ciliary neurotrophic factor (CNTF), indicating that they also act on astrocytes to promote axonal regrowth synergistically. These are the first data showing that axonal regeneration is related to crybb2 movement within neurons and to additional stimulation of CNTF. We demonstrate that neuronal crystallins constitute a novel class of neurite-promoting factors that probably operate through an autocrine and paracrine mechanism and that they can be used in neurodegenerative diseases. Thus, the post-injury fate of neurons cannot be seen merely as inevitable but, instead, must be regarded as a challenge to shape conditions for initiating growth cone formation to repair the damaged optic nerve.     

Cell type-specific Nogo-A gene ablation promotes axonal regeneration in the injured adult optic nerve

Nogo-A is a well-known myelin-enriched inhibitory protein for axonal growth and regeneration in the central nervous system (CNS). Besides oligodendrocytes, our previous data revealed that Nogo-A is also expressed in subpopulations of neurons including retinal ganglion cells, in which it can have a positive role in the neuronal growth response after injury, through an unclear mechanism. In the present study, we analyzed the opposite roles of glial versus neuronal Nogo-A in the injured visual system. To this aim, we created oligodendrocyte (Cnp-Cre^+/−xRtn4/Nogo-A^flox/flox) and neuron-specific (Thy1-Cre^tg+xRtn4^flox/flox) conditional Nogo-A knock-out (KO) mouse lines. Following complete intraorbital optic nerve crush, both spontaneous and inflammation-mediated axonal outgrowth was increased in the optic nerves of the glia-specific Nogo-A KO mice. In contrast, neuron-specific deletion of Nogo-A in a KO mouse line or after acute gene recombination in retinal ganglion cells mediated by adeno-associated virus serotype 2.Cre virus injection in Rtn4^flox/flox animals decreased axon sprouting in the injured optic nerve. These results therefore show that selective ablation of Nogo-A in oligodendrocytes and myelin in the optic nerve is more effective at enhancing regrowth of injured axons than what has previously been observed in conventional, complete Nogo-A KO mice. Our data also suggest that neuronal Nogo-A in retinal ganglion cells could participate in enhancing axonal sprouting, possibly by cis-interaction with Nogo receptors at the cell membrane that may counteract trans-Nogo-A signaling. We propose that inactivating Nogo-A in glia while preserving neuronal Nogo-A expression may be a successful strategy to promote axonal regeneration in the CNS.In the adult mammalian central nervous system (CNS), axons have a very limited capacity to regenerate after traumatic injury. This lack of axonal regeneration is thought to be mainly due to the presence of growth-inhibiting molecules in the injured CNS environment^{1, 2} and due to the low intrinsic growth capacity of mature neurons.³Nogo-A is a well-studied inhibitory protein for axonal growth, plasticity and regeneration after CNS injury.^{4, 5} Nogo-A is predominantly expressed in oligodendrocytes in the adult CNS, where it is thought to stabilize the neuronal circuits in healthy conditions and to inhibit neurite growth and plasticity after lesion.² Neutralizing Nogo-A by function-blocking antibodies or genetic knockout (KO) has been shown to improve axonal sprouting and regeneration in the injured spinal cord and brain.^{6, 7, 8, 9, 10, 11}In addition to oligodendrocytes and myelin, Nogo-A is expressed in growing and immature neurons, as well as in some adult neurons.^{12, 13} Neurons express Nogo-A receptors such as the Nogo-66 receptor 1 (NgR1)¹⁴ and the Nogo-A-Δ20-specific sphingosine 1-phosphate receptor 2 (S1PR2).¹⁵ They can co-express them along with Nogo-A,¹³ an observation that raises the possibility of cis-interactions between the ligand and its receptors within or at the cell surface of the same cell. This mechanism has previously been described for axonal guidance molecules such as Ephrins and Semaphorins, and could have a major role in the neuronal response to extracellular growth inhibitors during development.^{16, 17}In the adult CNS, the expression of neuronal Nogo-A remains elevated mainly in plastic regions such as in the hippocampus, olfactory bulb or neocortex, and in the dorsal root ganglia.¹² Nogo-A and NgR1 were shown to regulate synaptic plasticity, for example, long-term potentiation in the hippocampus and in the sensory-motor cortex,^{18, 19, 20, 21, 22} whereas the effects of neuronal Nogo-A after injury are not yet well understood. During development, neuronal Nogo-A influences neuronal migration,^{23, 24} survival,^{25, 26} cell spreading and neurite growth.^{27, 28} In injured adult retinal ganglion cells (RGCs), silencing neuronal Nogo-A resulted in a marked reduction of regenerative sprouting and decreased expression of growth-associated molecules.²⁹ Furthermore, in the optic nerve, axonal regeneration was not improved in conventional Nogo-A KO animals, in which both glial and neuronal Nogo-A were deleted.²⁹ The present study therefore aimed to investigate whether glial and neuronal Nogo-A differently influence axonal growth in vivo using cell type-specific Nogo-A KO mouse lines and adeno-associated virus (AAV)-mediated recombination of the Nogo-A gene in neurons. The results show that significantly more axons grew through the lesion site in the oligodendrocyte-specific Nogo-A KO mice. In contrast, neuron-specific ablation of Nogo-A in RGCs reduced the number of regenerating axons after optic nerve crush injury (ONC). In summary, we show that inactivating Nogo-A specifically in oligodendrocytes appears to be the most successful strategy to promote axonal regeneration in the adult optic nerve.     

Microfibril-microvessel connections in the uvea and optic nerve of the mammalian eye.

Light- and electron-microscopic examination of arterioles, venules and capillaries of the eyes of several mammalian species has shown that the microfibrils of ocular elastic tissue attach to microvascular basement membranes throughout the uvea (iris, ciliary body, choroid) and optic nerve. Although described sporadically in prior investigations, this report shows that the connections are a common feature of the mammalian eye. The connections appear most numerous at venules and capillaries and are sparse at arterioles. The connections may provide a mechanism by which perivascular elastic tissue influences microvascular function, e.g. the control of blood pressure in them or their response to changes in intraocular pressure.     

The differential appearance of neurofilament triplet polypeptides in the developing rat optic nerve

The ontogenetic appearance of the individual triplet polypeptides that comprise mammalian neurofilaments was studied in the developing rat optic nerve. Triton-insoluble cytoskeletal preparations from the optic nerves of rats of postnatal ages 1 Day (P1), 6 days (P6), 10 days (P10), 20 days (P20), and 3 months (adult) were analyzed for protein composition by one and two-dimensional gel electrophoresis. Results indicate that at P1, both the 150- and 68-kDa neurofilament subunit proteins are present. The 200-kDa subunit first becomes discernible at P20, but, at this age, it is still present in considerably less quantity than in the adult. Immunocytochemical verification of the presence of neurofilament protein was accomplished by staining tissue sections with specific antibodies against the 150- and the 68-kDa neurofilament subunits using the peroxidase-antiperoxidase technique. Results of the morphological analyses have shown that neurofilaments are not present in quantity until P10, which coincides with the time when the 68-kDa subunit increases in quantity by one dimensional gel analysis. Thus, the 150- and 68-kDa subunits can be detected prior to the appearance of neurofilaments, and the 200-kDa protein is not observed until sometime later. The potential physiological significance of the differential subunit transport is discussed with respect to neuronal differentiation in the developing mammalian CNS.     

Evidence that axon-derived neuregulin promotes oligodendrocyte survival in the developing rat optic nerve

It was previously shown that newly formed oligodendrocytes depend on axons for their survival, but the nature of the axon-derived survival signal(s) remained unknown. We show here that neuregulin (NRG) supports the survival of purified oligodendrocytes and aged oligodendrocyte precursor cells (OPCs) but not of young OPCs. We demonstrate that axons promote the survival of purified oligodendrocytes and that this effect is inhibited if NRG is neutralized. In the developing rat optic nerve, we provide evidence that delivery of NRG decreases both normal oligodendrocyte death and the extra oligodendrocyte death induced by nerve transection, whereas neutralization of endogenous NRG increases the normal death. These results suggest that NRG is an axon-associated survival signal for developing oligodendrocytes.     

Guidance of glial precursor cell migration by secreted cues in the developing optic nerve

Oligodendrocyte precursors are produced in restricted foci of the germinative neuroepithelium in embryo brains and migrate to their sites of function, while astrocytes are produced in a wider area in the neuroepithelium. We investigated the guidance mechanisms of glial precursor (GP) cell migration in the optic nerve. GP cell migration in newborn rat optic nerve was monitored by the UV-thymine-dimer (TD) method. A double labeling study using NG2 and TD revealed that many of these in vivo migrating cells were NG2 positive, while some of them with large TD-positive nuclei were NG2 negative. An in vitro cell migration study using optic nerve with chiasma and/or eyeball tissue revealed that the GP cells migrated under the guidance of repulsive cues secreted from the optic chiasma. We detected the expression of netrin 1 and Sema3a in the optic chiasma, and that of Unc5h1 and neuropilin 1 in the optic nerve. Co-culture experiments of the optic nerve with cell clusters expressing guidance cues revealed that the migrating GP cells in the optic nerve were heterogeneous. Netrin 1 repelled a subtype of NG2-positive and PLP-positive GP cells with small nuclei. Sema3a repelled a subtype of GP cells with large nuclei.     

Nitric oxide-cGMP signaling regulates axonal elongation during optic nerve regeneration in the goldfish in vitro and in vivo

Nitric oxide (NO) signaling results in both neurotoxic and neuroprotective effects in CNS and PNS neurons, respectively, after nerve lesioning. We investigated the role of NO signaling on optic nerve regeneration in the goldfish ( Carassius auratus ). NADPH diaphorase staining revealed that nitric oxide synthase (NOS) activity was up-regulated primarily in the retinal ganglion cells (RGCs) 5–40 days after axotomy. Levels of neuronal NOS (nNOS) mRNA and protein also increased in the RGCs alone during this period. This period (5–40 days) overlapped with the process of axonal elongation during regeneration of the goldfish optic nerve. Therefore, we evaluated the effect of NO signaling molecules upon neurite outgrowth from adult goldfish axotomized RGCs in culture. NO donors and dibutyryl cGMP increased neurite outgrowth dose-dependently. In contrast, a nNOS inhibitor and small interfering RNA, specific for the nNOS gene, suppressed neurite outgrowth from the injured RGCs. Intra-ocular dibutyryl cGMP promoted the axonal regeneration from injured RGCs in vivo . None of these molecules had an effect on cell death/survival in this culture system. This is the first report showing that NO-cGMP signaling pathway through nNOS activation is involved in neuroregeneration in fish CNS neurons after nerve lesioning.     

Akebia Saponin D prevents axonal loss against TNF-induced optic nerve damage with autophagy modulation

Akebia Saponin D (ASD), a triterpenoid saponin, was shown to have protective effects in certain neuronal cells. The purpose of the present study was to investigate the possibility of ASD to prevent tumor necrosis factor (TNF)-induced axonal loss and the ASD modulation of the biologic process of autophagy in optic nerves. Rats were given intravitreal administration of TNF, simultaneous administration of 2, 20, or 200 pmol ASD and TNF, or ASD alone. LC3-II and p62 expression, which is a marker of autophagic flux, and phosphorylated p38 (p-p38) expression in optic nerves were examined by immunoblot analysis. Morphometric analysis revealed a significant ameliorated effect of ASD against TNF-induced optic nerve damage. p62 was significantly increased in the optic nerve in TNF-treated eyes, but this increase was totally prevented by ASD. The ASD alone injection showed significant reduction of p62 levels compared with the PBS-treated control eyes. LC3-II was significantly increased by ASD treatment in the TNF-injected eyes. p-p38 was significantly increased in the optic nerve in TNF-treated eyes, but this increase was completely prevented by ASD. The protective effects of ASD may be associated with enhanced autophagy activation and inhibition of p-p38.

     

Neuritin 1 promotes retinal ganglion cell survival and axonal regeneration following optic nerve crush

Neuritin 1 (Nrn1) is an extracellular glycophosphatidylinositol-linked protein that stimulates axonal plasticity, dendritic arborization and synapse maturation in the central nervous system (CNS). The purpose of this study was to evaluate the neuroprotective and axogenic properties of Nrn1 on axotomized retinal ganglion cells (RGCs) in vitro and on the in vivo optic nerve crush (ONC) mouse model. Axotomized cultured RGCs treated with recombinant hNRN1 significantly increased survival of RGCs by 21% (n=6–7, P<0.01) and neurite outgrowth in RGCs by 141% compared to controls (n=15, P<0.05). RGC transduction with AAV2-CAG–hNRN1 prior to ONC promoted RGC survival (450%, n=3–7, P<0.05) and significantly preserved RGC function by 70% until 28 days post crush (dpc) (n=6, P<0.05) compared with the control AAV2-CAG–green fluorescent protein transduction group. Significantly elevated levels of RGC marker, RNA binding protein with multiple splicing (Rbpms; 73%, n=5–8, P<0.001) and growth cone marker, growth-associated protein 43 (Gap43; 36%, n=3, P<0.01) were observed 28 dpc in the retinas of the treatment group compared with the control group. Significant increase in Gap43 (100%, n=5–6, P<0.05) expression was observed within the optic nerves of the AAV2–hNRN1 group compared to controls. In conclusion, Nrn1 exhibited neuroprotective, regenerative effects and preserved RGC function on axotomized RGCs in vitro and after axonal injury in vivo. Nrn1 is a potential therapeutic target for CNS neurodegenerative diseases.Central nervous system (CNS) trauma and neurodegenerative disorders trigger a cascade of intrinsic and extrinsic cellular events resulting in regenerative failure and subsequent damage to neurons.^{1, 2, 3, 4, 5} The intrinsic factors include deregulation in growth-promoting factors, apoptotic factors, intracellular signaling molecules and trophic factors.⁶ Similarly, the extrinsic factors correlate to growth inhibition due to inhibitory cues^{3, 7, 8, 9, 10, 11, 12, 13} that include myelin and myelin associated inhibitors, glial scarring,^{5, 14} slow clearance of axonal debris,⁷ incorrect development of neuronal projections⁶ and CNS inflammation.^{15, 16} Progressive degeneration of mature retinal ganglion cells (RGCs) has been associated with loss of trophic support,^{8, 9} detrimental inflammatory processes/immune regulation^{10, 11} and apoptotic effectors.^{9, 12, 13, 15, 17}After injury, mammalian RGC axons show only a short-lived sprouting response but no long-distance regeneration through the optic nerve (ON).¹⁶ Glial responses around the affected area are initiated by injured CNS axons.¹⁸ Axons undergoing Wallerian degeneration are surrounded by astrocytes that upregulate glial fibrillary acidic protein (Gfap) expression and these reactive astrocytes contribute to trauma-induced neurodegeneration.¹⁹ Glial scarring inhibits axonal transport after ON crush (ONC)^{5, 14} decreasing transport of proteins involved in neuroprotection and synaptic plasticity. Regenerative failure is a critical endpoint of these destructive triggers culminating in neuronal apoptosis^{3, 20, 21} and inhibition of functional recovery. Intrinsic factors affecting axonal regeneration after CNS injury are crucial for recovery and thus, dysregulation of genes involved in axonal plasticity and outgrowth can prove detrimental to the neuronal recovery.^{22, 23, 24}Current neuroprotection approaches include promoting survival of RGCs by intraocular injections of recombinant factors like ciliary neurotrophic factor (CNTF) and peripheral nerve (PN) transplantations in vitro²⁵ and in vivo after injury.²⁶ Studies performed with glial cell-line-derived neurotrophic factor and neurturin protect RGCs from axotomy-induced apoptosis.²⁷ Further, in the ON injury model, RGC survival was promoted after deletion of CCAAT/enhancer binding protein homologous protein²⁸ and enhanced regeneration observed with co-deletion of kruppel-like factor 4 (Klf4) and suppressor of cytokine signaling 3 (Socs3).²⁹ Intraocular administration of neurotrophin-4 (NT-4) and brain-derived neurotrophic factor (BDNF) after ON transection has also exerted neuroprotective effects on axotomized RGCs. In addition, PNs transplanted adjacent to ONs, ex vivo PN grafts with lenti-viral transduced Schwann cells, and stimulation of inflammatory processes have strong pro-regenerative effects on injured RGCs.^{26, 30, 31, 32, 33}In addition, using adeno-associated-virus (AAV) therapy, AAV mediated expression of CNTF in bcl2 overexpressing transgenic mice increases cell viability and axonal regeneration,³⁴ whereas BDNF promotes survival of RGCs.³⁵ Likewise, experiments with AAV–BDNF, –CNTF and –growth-associated protein 43 (GAP43) have shown that AAV–CNTF was the most crucial for promoting both long-term survival and regeneration.³⁶ The positive effects of CNTF are observed mainly through simultaneous deletion of both PTEN and SOCS3³⁷ and the concurrent activation of mTOR and STAT3 pathways.³⁸ Although CNTF shows robust increase and sustained axon regeneration in injured ONs of rodents, it causes axonal misguidance and aberrant growth.³⁹ Furthermore, it has been shown that CNTF acts as a chemoattractant. CNTF administration onto autologous PN grafts transplanted within transected ON increased regeneration, but these effects were significantly reduced after removal of macrophages from this site.⁴⁰ In addition, the effects of CNTF using PN grafts at ON transection sites are further subject to debate, as previously it has been shown that Ad-CNTF injections preserved RGC axons but did not induce regeneration of axotomized RGCs.⁴¹ Thus, other studies have addressed RGC survivability and axonal regeneration with CNTF and other growth factors,^{35, 36} but most trophic factors affect neuronal survival and regeneration differentially.Previous studies targeting neuronal apoptosis by overexpressing intrinsic growth factors, inhibiting apoptosis and enhancing regeneration in CNS trauma models have established that a multifactorial approach is required for successful and long-lasting therapeutic outcomes.^{6, 36} Current gaps still exist for a key gene that could effectively target neuroprotection, enhance neuron regeneration and sustain neuronal function.One key gene implicated in neuronal plasticity is Neuritin 1 (Nrn1), also known as candidate plasticity gene 15. It has multiple functions and was first identified and characterized when screening for candidate plasticity genes in the rat hippocampal dentate gyrus activated by kainate.^{42, 43, 44} Nrn1 is highly conserved across species⁴⁵ and translates to an extracellular, glycophosphatidylinositol-linked protein (GPI-linked protein), which can be secreted as a soluble form. Nrn1 stimulates axonal plasticity, dendritic arborization and synapse maturation in the CNS.⁴⁶ During early embryonic development, Nrn1 promotes the survival of neural progenitors and differentiated neurons,⁴⁷ while later in development it promotes axonal and dendritic growth and stabilization, allowing maturation and formation of synapses.^{43, 46, 48} In the adult brain, Nrn1 has been correlated with activity-dependent functional plasticity^{45, 49} and is expressed in post mitotic neurons.Nrn1 may be a crucial gene for neuroprotection and regeneration because growth factors such as nerve growth factor (NGF), BDNF and NT-3 as well as neuronal activity can potentiate the expression of Nrn1.^{44, 50} In addition, we reported that Nrn1 mRNA expression appears to be biphasic after ON axonal trauma, indicating a transient attempt by RGCs at neuroprotection/neuroregeneration in response to ONC injury.⁵¹ The dynamic regulation of Nrn1 coupled with neurotrophic effects may promote axonal regeneration in the CNS. To overcome CNS trauma, a new therapy geared towards neuroprotection and effective axonal regeneration is required to enhance a future multifactorial approach. The purpose of this study is to evaluate the therapeutic effects of Nrn1 in mouse RGC cultures as well as in the mouse ONC model. We have identified a distinct neuroprotective and regenerative strategy that prevents neurodegeneration after ON injury. AAV2–hNRN1 expression vectors partially rescued RGCs from apoptosis, maintained RGC function, and initiated regeneration of injured axons.     

K+ channel distribution and clustering in developing and hypomyelinated axons of the optic nerve

The localization of Shaker-type K⁺ channels in specialized domains of myelinated central nervous system axons was studied during development of the optic nerve. In adult rats Kv1.1, Kv1.2, Kv1.6, and the cytoplasmic β-subunit Kvβ2 were colocalized in juxtaparanodal zones. During development, clustering of K⁺ channels lagged behind that for nodal Na⁺ channels by about 5 days. In contrast to the PNS, K⁺ channels were initially expressed fully segregated from nodes and paranodes, the latter identified by immunofluorescence of Caspr, a component of axoglial junctions. Clusters of K⁺ channels were first detected at postnatal day 14 (P14) at a limited number of sites. Expression increased until all juxtaparanodes had immunoreactivity by P40. Developmental studies in hypomyelinating Shiverer mice revealed dramatically disrupted axoglial junctions, aberrant Na⁺ channel clusters, and little or no detectable clustering of K⁺ channels at all ages. These results suggest that in the optic nerve, compact myelin and normal axoglial junctions are essential for proper K⁺ channel clustering and localization.     

A semiautomated targeted sampling method to assess optic nerve axonal loss in a rat model of glaucoma

We have developed a fast, reliable and easily reproducible semiautomated quantitative damage grading scheme to assess axonal loss in the optic nerve after inducing ocular hypertension using a laser glaucoma model in adult rats. This targeted sampling method has been validated against complete axon counts, and compares favorably with a conventional, random sampling, semiquantitative method. In addition, we present a standardized method to quantify axons in a semiautomated way, using freely available ImageJ software, and describe in detail the method used to induce glaucoma. Our techniques can be easily implemented in any laboratory, thanks to the public availability of the software and the simplicity of the method. Depending on the number of animals used in a particular study, the whole process from experimental elevation of intraocular pressure to tissue processing and data analysis should take ～40 d.     

Misguidance and modulation of axonal regeneration by Stat3 and Rho/ROCK signaling in the transparent optic nerve

The use of the visual system played a major role in the elucidation of molecular mechanisms controlling axonal regeneration in the injured CNS after trauma. In this model, CNTF was shown to be the most potent known neurotrophic factor for axonal regeneration in the injured optic nerve. To clarify the role of the downstream growth regulator Stat3, we analyzed axonal regeneration and neuronal survival after an optic nerve crush in adult mice. The infection of retinal ganglion cells with adeno-associated virus serotype 2 (AAV2) containing wild-type (Stat3-wt) or constitutively active (Stat3-ca) Stat3 cDNA promoted axonal regeneration in the injured optic nerve. Axonal growth was analyzed in whole-mounted optic nerves in three dimensions (3D) after tissue clearing. Surprisingly, with AAV2.Stat3-ca stimulation, axons elongating beyond the lesion site displayed very irregular courses, including frequent U-turns, suggesting massive directionality and guidance problems. The pharmacological blockade of ROCK, a key signaling component for myelin-associated growth inhibitors, reduced axonal U-turns and potentiated AAV2.Stat3-ca-induced regeneration. Similar results were obtained after the sustained delivery of CNTF in the axotomized retina. These results show the important role of Stat3 in the activation of the neuronal growth program for regeneration, and they reveal that axonal misguidance is a key limiting factor that can affect long-distance regeneration and target interaction after trauma in the CNS. The correction of axonal misguidance was associated with improved long-distance axon regeneration in the injured adult CNS.