Extracellular signal-regulated kinase 1/2 mediates survival, but not axon regeneration, of adult injured central nervous system neurons in vivo

Neurotrophins play important roles in the response of adult neurons to injury. The intracellular signaling mechanisms used by neurotrophins to regulate survival and axon growth in the mature CNS in vivo are not well understood. The goal of this study was to define the role of the extracellular signal-regulated kinases 1/2 (Erk1/2) pathway in the survival and axon regeneration of adult rat retinal ganglion cells (RGCs), a prototypical central neuron population. We used recombinant adeno-associated virus (AAV) to selectively transduce RGCs with genes encoding constitutively active or wild-type mitogen-activated protein kinase kinase 1 (MEK1), the upstream activator of Erk1/2. In combination with anterograde and retrograde tracing techniques, we monitored neuronal survival and axon regeneration in vivo. MEK1 gene delivery led to robust and selective transgene expression in multiple RGC compartments including cell bodies, dendrites, axons and targets in the brain. Furthermore, MEK1 activation induced in vivo phosphorylation of Erk1/2 in RGC bodies and axons. Quantitative analysis of cell survival demonstrated that Erk1/2 activation promoted robust RGC neuroprotection after optic nerve injury. In contrast, stimulation of the Erk1/2 pathway was not sufficient to induce RGC axon growth beyond the lesion site. We conclude that the Erk1/2 pathway plays a key role in the survival of axotomized mammalian RGCs in vivo, and that activation of other signaling components is required for axon regeneration in the growth inhibitory CNS environment.     

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.     

Upregulation of IGF-I in the goldfish retinal ganglion cells during the early stage of optic nerve regeneration

Goldfish retinal ganglion cells (RGCs) can regrow their axons after optic nerve injury. However, the reason why goldfish RGCs can regenerate after nerve injury is largely unknown at the molecular level. To investigate regenerative properties of goldfish RGCs, we divided the RGC regeneration process into two components: (1) RGC survival, and (2) axonal elongation processes. To characterize the RGC survival signaling pathway after optic nerve injury, we investigated cell survival/death signals such as Bcl-2 family members in the goldfish retina. Amounts of phospho-Akt (p-Akt) and phospho-Bad (p-Bad) in the goldfish retina rapidly increased four- to five-fold at the protein level by 3-5 days after nerve injury. Subsequently, Bcl-2 levels increased 1.7-fold, accompanied by a slight reduction in caspase-3 activity 10-20 days after injury. Furthermore, level of insulin-like growth factor-I (IGF-I), which activates the phosphatidyl inositol-3-kinase (PI3K)/Akt system, increased 2-3 days earlier than that of p-Akt in the goldfish retina. The cellular localization of these molecular changes was limited to RGCs. IGF-I treatment significantly induced phosphorylation of Akt, and strikingly induced neurite outgrowth in the goldfish retina in vitro. On the contrary, addition of the PI3K inhibitor wortmannin, and IGF-I antibody inhibited Akt phosphorylation and neurite outgrowth in an explant culture. Thus, we demonstrated, for the first time, the signal cascade for early upregulation of IGF-I, leading to RGC survival and axonal regeneration in adult goldfish retinas through PI3K/Akt system after optic nerve injury. The present data strongly indicate that IGF-I is one of the most important molecules for controlling regeneration of RGCs after optic nerve injury.     

Inhibition of Rho kinase (ROCK) increases neurite outgrowth on chondroitin sulphate proteoglycan in vitro and axonal regeneration in the adult optic nerve in vivo

Inhibitory molecules derived from CNS myelin and glial scar tissue are major causes for insufficient functional regeneration in the mammalian CNS. A multitude of these molecules signal through the Rho/Rho kinase (ROCK) pathway. We evaluated three inhibitors of ROCK, Y- 27632, Fasudil (HA-1077), and Dimethylfasudil (H-1152), in models of neurite outgrowth in vitro. We show, that all three ROCK inhibitors partially restore neurite outgrowth of Ntera-2 neurons on the inhibitory chondroitin sulphate proteoglycan substrate. In the rat optic nerve crush model Y-27632 dose-dependently increased regeneration of retinal ganglion cell axons in vivo. Application of Dimethylfasudil showed a trend towards increased axonal regeneration in an intermediate concentration. We demonstrate that inhibition of ROCK can be an effective therapeutic approach to increase regeneration of CNS neurons. The selection of a suitable inhibitor with a broad therapeutic window, however, is crucial in order to minimize unwanted side effects and to avoid deleterious effects on nerve fiber growth.     

Early downregulation of IGF-I decides the fate of rat retinal ganglion cells after optic nerve injury

Retinal ganglion cells (RGCs) die by apoptosis after optic nerve injury. A number of reports have separately shown changes in pro-apoptotic proteins such as the Bcl-2 family members following optic nerve injury. However, induction time of these apoptotic signals has not been identified due to different treatments of the optic nerve, and insufficient time intervals for measurements. Therefore, the stream of cell death signals is not well understood. In the present study, we systematically reinvestigated a detailed time course of these cell death/survival signals in the rat retina after optic nerve crush, to determine the signal cascade leading to RGC apoptosis. The most conspicuous changes detected in the retina were the rapid inactivation of phospho-Akt and phospho-Bad proteins 2-3 days after optic nerve damage, and the subsequent gradual activation of Bax protein and caspase-3 activity accompanied by cell loss of RGCs 6 days after nerve injury. Cellular localization of these molecular changes was limited to RGCs. Furthermore, amount of insulin-like growth factor-I (IGF-I), an activator of the phosphatidyl inositol-3-kinase (PI3K)/Akt system, was initially decreased from RGCs 1-2 days just prior to the inactivation of phospho-Akt by optic nerve crush. Conversely, supplementation with IGF-I into the rat retina induced upregulation of phospho-Akt expression and cell survival of RGCs both in vitro and in vivo. Thus, injury to the optic nerve might induce early changes in cellular homeostasis with a plausible loss of trophic support for injured RGCs. Actually, IGF-I drastically enhanced neurite outgrowth from adult rat RGCs via a wortmannin-dependent mechanism in a retinal explant culture. Our data strongly indicate that IGF-I is a key molecule that induces RGC apoptosis or RGC survival and regeneration in the retina during the early stage of optic nerve injury.     

Quantitative iTRAQ analysis of retinal ganglion cell degeneration after optic nerve crush

Retinal ganglion cells (RGCs) are central nervous system (CNS) neurons that transmit visual information from the retina to the brain. Apoptotic RGC degeneration causes visual impairment that can be modeled by optic nerve crush. Neuronal apoptosis is also a salient feature of CNS trauma, ischemia (stroke), and diseases of the CNS such as Alzheimer's, Parkinson's, multiple sclerosis, and amyotrophic lateral sclerosis. Optic nerve crush induces the apoptotic cell death of ～ 70% of RGCs within the first 14 days after injury. This model is particularly attractive for studying adult neuron apoptosis because the time-course of RGC death is well established and axon regeneration within the myelinated optic nerve can be concurrently evaluated. Here, we performed a large scale iTRAQ proteomic study to identify and quantify proteins of the rat retina at 1, 3, 4, 7, 14, and 21 days after optic nerve crush. In total, 337 proteins were identified, and 110 were differentially regulated after injury. Of these, 58 proteins were upregulated (>1.3 ×), 46 were downregulated (<0.7 ×), and 6 showed both positive and negative regulation over 21 days, relative to normal retinas. Among the differentially expressed proteins, Thymosin-β4 showed an early upregulation at 3 days, the time-point that immediately precedes the induction of RGC apoptosis after injury. We examined the effect of exogenous Thymosin-β4 administration on RGC death after optic nerve injury. Intraocular injections of Thymosin-β4 significantly increased RGC survival by ～ 3-fold compared to controls and enhanced axon regeneration after crush, demonstrating therapeutic potential for CNS insults. Overall, our study identified numerous proteins that are differentially regulated at key time-points after optic nerve crush, and how the temporal profiles of their expression parallel RGC death. This data will aid in the future development of novel therapeutics to promote neuronal survival and regeneration in the adult CNS.     

Neuronal Nogo-A upregulation does not contribute to ER stress-associated apoptosis but participates in the regenerative response in the axotomized adult retina

Nogo-A, an axonal growth inhibitory protein known to be mostly present in CNS myelin, was upregulated in retinal ganglion cells (RGCs) after optic nerve injury in adult mice. Nogo-A increased concomitantly with the endoplasmic reticulum stress (ER stress) marker C/EBP homologous protein (CHOP), but CHOP immunostaining and the apoptosis marker annexin V did not co-localize with Nogo-A in individual RGC cell bodies, suggesting that injury-induced Nogo-A upregulation is not involved in axotomy-induced cell death. Silencing Nogo-A with an adeno-associated virus serotype 2 containing a short hairpin RNA (AAV2.shRNA-Nogo-A) or Nogo-A gene ablation in knock-out (KO) animals had little effect on the lesion-induced cell stress or death. On the other hand, Nogo-A overexpression mediated by AAV2.Nogo-A exacerbated RGC cell death after injury. Strikingly, however, injury-induced sprouting of the cut axons and the expression of growth-associated molecules were markedly reduced by AAV2.shRNA-Nogo-A. The axonal growth in the optic nerve activated by the intraocular injection of the inflammatory molecule Pam3Cys tended to be lower in Nogo-A KO mice than in WT mice. Nogo-A overexpression in RGCs in vivo or in the neuronal cell line F11 in vitro promoted regeneration, demonstrating a positive, cell-autonomous role for neuronal Nogo-A in the modulation of axonal regeneration.     

Promoting Optic Nerve Regeneration in Adult Mice with Pharmaceutical Approach

Our previous research has suggested that lack of Bcl-2-supported axonal growth mechanisms and the presence of glial scarring following injury are major impediments of optic nerve regeneration in postnatal mice. Mice overexpressing Bcl-2 and simultaneously carrying impairment in glial scar formation supported robust optic nerve regeneration in the postnatal stage. To develop a therapeutic strategy for optic nerve damage, the combined effects of chemicals that induce Bcl-2 expression and selectively eliminate mature astrocytes—scar forming cells—were examined in mice. Mood-stabilizer, lithium, has been shown to induce Bcl-2 expression and stimulate axonal outgrowth in retinal ganglion cells in culture and in vivo. Moreover, astrotoxin (alpha-aminoadipate), a glutamate analogue, selectively kills astrocytes while has minimal effects on surrounding neurons. In the present study, we sought to determine whether concurrent applications of lithium and astrotoxin were sufficient to induce optic nerve regeneration in mice. Induction of Bcl-2 expression was detected in the ganglion cell layer (GCL) of mice that received a lithium diet in compared with control-treated group. Moreover, efficient elimination of astrocytes and glial scarring was observed in the optic nerve of mice treated with astrotoxin. Simultaneous application of lithium and astrotoxin, but not any of the drugs alone, induced robust optic nerve regeneration in adult mice. These findings further support that a combinatorial approach of concurrent activation of Bcl-2-supported growth mechanism and suppression of glial scarring is required for successful regeneration of the severed optic nerve in adult mice. They suggest a potential therapeutic strategy for treating optic nerve and CNS damage. Special issue article in honor of Dr. Ji-Sheng Han.     

Laminin-immunoreactive glia distinguish regenerative adult CNS systems from non-regenerative ones.

Most regions of the adult mammalian central nervous system (CNS) do not support axonal growth and regeneration. Laminin, expressed by cultured astrocytes and known to promote neurite outgrowth of cultured neurons, is normally present in brain basement membranes, and only transiently induced in adult brain astrocytes by injury. Here I provide three lines of evidence which suggest that the continued expression of laminin by astrocytes may be a prerequisite for axonal growth and regeneration in adult CNS. Firstly, laminin is continuously present in astrocytes of adult rat olfactory bulb apparently in close association with the olfactory nerve axons. Secondly, laminin is continuously expressed by astrocytes in adult frog brain, and sectioning of the optic tract further increases laminin immunoreactivity in astrocytes of the optic tectum during the period of axonal regeneration. Lastly, laminin appears normally in astrocytes of the frog and goldfish optic nerves which regenerate, but not in astrocytes of the rat or chick optic nerves which do not regenerate. The selective association of laminin with axons that undergo growth and regeneration in vivo is consistent with the possibility that astrocytic laminin provides these central nervous systems with their regenerative potential.     

Differential expression of calretinin in the developing and regenerating zebrafish visual system

Calretinin is a calcium-binding protein which participates in a variety of functions including calcium buffering and neuronal protection. It also serves as a developmental marker of retinal ganglion cells (RGCs). In order to study the role of calretinin in the development and regeneration of RGCs, we have studied its pattern of expression in the retina at different developmental stages, as well as during optic nerve regeneration by means of immunohistochemistry. During development, calretinin is found for the first time in RGCs when they connect with the optic tectum. Optic nerves from adult zebrafish were crushed and after different survival times, calretinin expression in the retina, optic nerve tract and optic tectum was studied. From the day of crushing to 10 days later, calretinin expression was found to be downregulated within RGCs and their axons, as was also observed during the early developmental stages of RGCs, when they are not committed to a definite cell phenotype. Moreover, 13 days after lesion, when the regenerating axons arrived at the optic tectum, a recovery of calretinin immunoreactivity within the RGCs was observed. These results indicate that calretinin may play an important role during optic nerve regeneration, Thus, the down-regulation of Calretinin during the growth of the RGC axons towards the target during development as well as during their regeneration after injury, indicates that an increase the availability of cytosolic calcium is integral to axon outgrowth thus recapitulating the pattern observed during development.     

Hepatocyte growth factor is necessary for efficient outgrowth of injured peripheral axons in in vitro culture system and in vivo nerve crush mouse model

Hepatocyte growth factor (HGF) is a neurotrophic factor and its role in peripheral nerves has been relatively unknown. In this study, biological functions of HGF and its receptor c-met have been investigated in the context of regeneration of damaged peripheral nerves. Axotomy of the peripheral branch of sensory neurons from embryonic dorsal root ganglia (DRG) resulted in the increased protein levels of HGF and phosphorylated c-met. When the neuronal cultures were treated with a pharmacological inhibitor of c-met, PHA665752, the length of axotomy-induced outgrowth of neurite was significantly reduced. On the other hand, the addition of recombinant HGF proteins to the neuronal culture facilitated axon outgrowth. In the nerve crush mouse model, the protein level of HGF was increased around the injury site by almost 5.5-fold at 24 h post injury compared to control mice and was maintained at elevated levels for another 6 days. The amount of phosphorylated c-met receptor in sciatic nerve was also observed to be higher than control mice. When PHA665752 was locally applied to the injury site of sciatic nerve, axon outgrowth and injury mediated induction of cJun protein were effectively inhibited, indicating the functional involvement of HGF/c-met pathway in the nerve regeneration process. When extra HGF was exogenously provided by intramuscular injection of plasmid DNA expressing HGF, axon outgrowth from damaged sciatic nerve and cJun expression level were enhanced. Taken together, these results suggested that HGF/c-met pathway plays important roles in axon outgrowth by directly interacting with sensory neurons and thus HGF might be a useful tool for developing therapeutics for peripheral neuropathy.     

GABA and development of the Xenopus optic projection

In the developing visual system of Xenopus laevis retinal ganglion cell (RGC) axons extend through the brain towards their major target in the midbrain, the optic tectum. Enroute, the axons are guided along their pathway by cues in the environment. In vitro, neurotransmitters have been shown to act chemotropically to influence the trajectory of extending axons and regulate the outgrowth of developing neurites, suggesting that they may act to guide or modulate the growth of axons in vivo. Previous work by Roberts and colleagues (1987) showed that populations of cells within the developing Xenopus diencephalon and mid-brain express the neurotransmitter gamma amino butyric acid (GABA). Here we show that Xenopus RGC axons in the midoptic tract grow alongside the GABAergic cells and cross their GABA immunopositive nerve processes. Moreover, RGC axons and growth cones express GABA-A and GABA-B receptors, and GABA and the GABA-B receptor agonist baclofen both stimulate RGC neurite outgrowth in culture. Finally, the GABA-B receptor antagonist CGP54626 applied to the developing optic projection in vivo causes a dose-dependent shortening of the optic projection. These data indicate that GABA may act in vivo to stimulate the outgrowth of Xenopus RGC axons along the optic tract.     

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.     

Arterial cells and CNS sheath cells from Aplysia californica produce factors that enhance neurite outgrowth in co-cultured neurons

Substrate-bound and soluble factors regulate neurite outgrowth and synapse formation during development, regeneration, and learning and memory. We report that sheath cells from CNS connectives and arterial cells from the anterior aorta of the sea slug, Aplysia californica, enhance neurite outgrowth from co-cultured Aplysia neurons. Sheath and arterial cell cultures contain several cell types, including fibrocytes, myocytes, and amoebocytes. When compared to controls (neurons with defined growth medium alone), the percentage of neurons with growth and the average neurite lengths are significantly enhanced by sheath and arterial cells at 48 h after plating of the neurons; these parameters are comparable to those of neurons cultured in medium containing hemolymph. Our results indicate that sheath cells produce substrate-bound factor(s) and arterial cells produce diffusible factor(s) that promote growth. These growth factors likely promote neuron survival and neurite outgrowth during neural plasticity exhibited in the adult CNS. Electronic Publication     

Retinoic acid induces neurite outgrowth and growth cone turning in invertebrate neurons

Identification of molecules involved in neurite outgrowth during development and/or regeneration is a major goal in the field of neuroscience. Retinoic acid (RA) is a biologically important metabolite of vitamin A that acts as a trophic factor and has been implicated in neurite outgrowth and regeneration in many vertebrate species. Although abundant in the CNS of many vertebrates, the precise role of RA in neural regeneration has yet to be determined. Moreover, very little information is available regarding the role of RA in invertebrate nervous systems. Here, we demonstrate for the first time that RA induces neurite outgrowth from invertebrate neurons. Using individually identified neurons isolated from the CNS of Lymnaea stagnalis, we demonstrated that a significantly greater proportion of cells produced neurite outgrowth in RA. RA also extended the duration of time that cells remained electrically excitable in vitro, and we showed that exogenously applied RA acted as a chemoattractive factor and induced growth cone turning toward the source of RA. This is the first demonstration that RA can induce turning of an individual growth cone. These data strongly suggest that the actions of RA on neurite outgrowth and cell survival are highly conserved across species.     

Activation of HGF/c-Met signaling by ultrafine carbon particles and its contribution to alveolar type II cell proliferation

Hepatocyte growth factor (HGF) is a potent mitogen and motogen for various epithelial cells. The present study aimed to explore the role of HGF and c-Met receptor in ultrafine carbon particle-induced alveolar type II epithelial (type II) cell proliferation. ICR mice were intratracheally instilled with 100 μg ultrafine carbon black (ufCB) and killed at 21, 48, and 72 days postexposure to examine type II cell proliferation, HGF release, and c-Met activation. In vivo and in vitro applications of neutralizing anti-HGF antibody were used to investigate the causal role of HGF in cell proliferation. The Met kinase inhibitor SU11274 and extracellular signal-regulated kinase 1/2 (ERK1/2) inhibitor PD98059 were used to delineate the involvement of c-Met/ERK1/2 in rat L2 pulmonary epithelial cell proliferation. The results demonstrated that in vivo exposure to 100 μg ufCB caused increased HGF in bronchoalveolar lavage fluid, as well as increased HGF production, c-Met phosphorylation, and cell proliferation in type II cells. In vitro study revealed that ufCB caused a dose-dependent increase in HGF release, c-Met phosphorylation, and cell proliferation. Importantly, treatment with the neutralizing anti-HGF antibody significantly blocked ufCB-induced in vivo and in vitro type II cell proliferation. Moreover, SU11274 and PD98059 significantly reduced ufCB-increased L2 cell proliferation. Results from Western blotting demonstrated that SU11274 successfully suppressed ufCB-induced phosphorylation of c-Met and ERK1/2. In summary, the activation of HGF/c-Met signaling is a major pathway involved in ufCB-induced type II cell proliferation.     

Requirement of Retinoic Acid Receptor β for Genipin Derivative-Induced Optic Nerve Regeneration in Adult Rat Retina

Like other CNS neurons, mature retinal ganglion cells (RGCs) are unable to regenerate their axons after nerve injury due to a diminished intrinsic regenerative capacity. One of the reasons why they lose the capacity for axon regeneration seems to be associated with a dramatic shift in RGCs’ program of gene expression by epigenetic modulation. We recently reported that (1R)-isoPropyloxygenipin (IPRG001), a genipin derivative, has both neuroprotective and neurite outgrowth activities in murine RGC-5 retinal precursor cells. These effects were both mediated by nitric oxide (NO)/S-nitrosylation signaling. Neuritogenic activity was mediated by S-nitrosylation of histone deacetylase-2 (HDAC2), which subsequently induced retinoic acid receptor β (RARβ) expression via chromatin remodeling in vitro. RARβ plays important roles of neural growth and differentiation in development. However, the role of RARβ expression during adult rat optic nerve regeneration is not clear. In the present study, we extended this hypothesis to examine optic nerve regeneration by IPRG001 in adult rat RGCs in vivo. We found a correlation between RARβ expression and neurite outgrowth with age in the developing rat retina. Moreover, we found that IPRG001 significantly induced RARβ expression in adult rat RGCs through the S-nitrosylation of HDAC2 processing mechanism. Concomitant with RARβ expression, adult rat RGCs displayed a regenerative capacity for optic axons in vivo by IPRG001 treatment. These neuritogenic effects of IPRG001 were specifically suppressed by siRNA for RARβ. Thus, the dual neuroprotective and neuritogenic actions of genipin via S-nitrosylation might offer a powerful therapeutic tool for the treatment of RGC degenerative disorders.     

A primary culture technique of adult retina for regeneration studies on adult CNS neurons

This protocol details a tissue culture technique that allows for quantified regeneration studies on adult retinal ganglion cells (RGCs), that is, CNS neurons. The method may also allow for elucidation of molecular cues, for example of signals relevant in neuronal survival and axon regeneration. The procedure relies on fractioned stripe culture of previously injured retina in defined culture media. Naive dendritic cell contacts of RGCs are preserved, and the system is independent of growth factors. In contrast to other techniques, the protocol is based on tissue grown from adult animals; it dispenses immature co-cultures and evaluates the outgrowth of unmyelinated neurites in a milieu lacking CNS myelin. The technique is suitable for rodent retina from mouse or rat. A growth-conditioning injury of the optic nerve is set 10 days before retinal explantation. Explants are cultured for 5-7 days. Mere preparation of a single retina should be completed within 20 min.     

Guidance of regenerating motor axons in vivo by gradients of diffusible peripheral nerve-derived factors

During development of the central nervous system, neurons rely on target-derived factors to guide their outgrowing processes. Several CNS target-derived chemoattractive and repellent factors have been isolated and characterized, and their mechanism of action determined. For the peripheral nervous system, the results from numerous experiments suggest that during regeneration axons also respond to concentration gradients of target-derived factors leading to an oriented outgrowth up the gradient to the denervated target in vivo. The results from in vitro experiments have shown that diffusible concentration gradients of factors released from a length of denervated peripheral nerve, composed predominantly of Schwann cells, direct the outgrowth of sensory and motor neuron growth cones over distances of several hundred microns. However, a conclusive demonstration of a chemoattractive influence of diffusible concentration gradients on regenerating adult motor axons in vivo has remained elusive. The present experiments show that concentration gradients of denervated peripheral nerve-released factors direct the regeneration of adult motor axons in vivo, and that these gradients are effective over distances of more than 6.5 mm. Nonconditioned medium exerted no influence on the regenerating axons. Thus, results from in vivo experiments parallel those from in vitro experiments and indicate that isolated peripheral nerve-released factors that are effective in vitro will play a similar role on sensory and motor axons in vivo. Finally, the results show that diffusible concentration gradients of target-derived factors direct axon outgrowth both during both development and regeneration, as well as in vivo and in vitro.     

Molecular and cellular aspects of axon-glia interaction in CNS regeneration

The relationships of neurons and non-neuronal cells are vital for the maintenance and function of neurons. Trauma alters these relationships causing proliferation of non-neuronal cells and, in adult mammalian CNS, presumably disturbs the environmental support needed for regeneration. A supportive environment can be restored by introducing a regenerating nerve to injured mammalian CNS. This response is probably due, at least in part, to diffusible substances secreted by the non-neuronal cells. We have obtained diffusible substances from either regenerating fish optic nerves or neonatal rabbit optic nerves and applied them around crushed adult rabbit optic nerves. This manipulation caused the adult nerve to show regenerative changes: a general increase of protein synthesis in the retinas; selective increase in synthesis of a few polypeptides in the retinas; sprouting from the retinas in vitro; increased viability of nerve fibers as shown by HRP staining; and the appearance of growth cones adjacent to glial limitans in the injured nerves. We termed these diffusible, active substances "Growth Associated Triggering Factors" (GATFs). In addition to the phenomena described above, the active substances (obtained in the form of media conditioned by regenerating fish optic nerve or neonatal rabbit optic nerve) caused various other changes in the injured nerve itself: acceleration of non-neuronal cell proliferation; changes in the protein pattern, e.g. an increase in a 12 kDa polypeptide which might be a second mediator in the cascade of events leading to regeneration; increased laminin immunoreactive sites in the nerve; and the acquisition of growth supportive activity in media conditioned by the implanted injured nerves.(ABSTRACT TRUNCATED AT 250 WORDS)