Schwann cell surfaces but not extracellular matrix organized by Schwann cells support neurite outgrowth from embryonic rat retina

Despite evidence that glial cell surfaces and components of the extracellular matrix (ECM) support neurite outgrowth in many culture systems, the relative contributions of these factors have rarely been compared directly. Specifically, it remains to be determined which components of peripheral nerve support growth of central nerve fibers. We have directly compared neurite outgrowth from embryonic day 15 rat retinal explants placed onto beds of (1) Schwann cells without ECM, (2) Schwann cells expressing ECM (including a basal lamina), (3) cell-free ECM prepared from neuron-Schwann cell cultures, (4) nonglial cells (fibroblasts), and (5) 2 isolated ECM components, laminin and type I collagen. From the first day in culture, retinal explants extended neurites when placed on Schwann cells without ECM. Outgrowth on Schwann cells expressing ECM was also extensive, but not obviously different form that on Schwann cells alone. Ultrastructural study revealed that 95% of retinal neurites in ECM-containing cultures contacted other neurites and Schwann cell surfaces exclusively. On cell-free ECM prepared from neuron-Schwann cell cultures, neurite extension was poor to nonexistent. No neurite outgrowth occurred on fibroblasts. Retinal explants also failed to extend neurites onto purified laminin and ammoniated type I collagen substrata; however, growth was rapid and extensive on air-dried type I collagen. In cultures containing islands of air-dried type I collagen on a laminin-coated coverslip, retinal explants attached and extended neurites on collagen, but these neurites did not extend off the island onto the laminin substratum. We conclude from these experiments that neurite extension from embryonic rat retina is supported by a factor found on the surface of Schwann cells and that neither organized nor isolated ECM components provide this neurite promotion. These findings are discussed in relation to possible species differences in growth requirements for retinal ganglion cell neurites and to the specificity of response of different CNS neurites to ECM substrata.     

Functional status influences the ability of Schwann cells to support adult rat retinal ganglion cell survival and axonal regrowth

Properties of Schwann cells (SC) within the environment of the peripheral nerve can differ depending on their state of differentiation (e.g., quiescent, proliferating or mature SCs). In the present study we have tested the efficacy of SCs in different functional states to promote adult rat retinal ganglion cell (RGC) survival and neurite growth. We have used culture conditions which allowed regenerating retinal axons to contact SCs (1) as quiescent SCs that had not been in contact with axons, (2) as SCs that had been proliferating in contact with neurites, and (3) as mature SCs which had myelinated axons and deposited basal lamina around the axon-SC unit. Both proliferating and mature SCs were activated, i.e., were removed from axonal contact by inducing Wallerian degeneration, 2-3 days prior to testing their ability to support neuronal survival and neurite growth. Activated SCs, (derived from either proliferating or mature SCs) supported adult rat RGC survival and axonal regrowth significantly better than quiescent SC monolayers. Conditioned media (CM) derived from corresponding SC preparations (quiescent, proliferating, or mature SCs) supported short-term survival of RGCs and neurite outgrowth on defined substrata. A dissociation of substrate adsorbable neurite-promoting and nonadsorbable neurotrophic factors could be observed. The neurite-promoting activities found in CM were trypsin and heat sensitive, suggesting that the active component(s) is protein(s). None of the CM tested was able to support adult rat RGC survival and axonal growth as effectively as activated SCs (either proliferating or mature SCs). Long-term survival (greater than 4 days) of RGCs in explant cultures were especially dependent on RGC-neurite contact with preactivated SCs; CM derived from corresponding SCs (mature and proliferating SC-CM) were significantly less effective in supporting long-term survival. From these data we conclude that consideration must be given to the functional state of the SCs in experiments designed to utilize SCs in promoting CNS regeneration.     

Adult rat olfactory nerve ensheathing cells are effective promoters of adult central nervous system neurite outgrowth in coculture

A coculture method is described for ensheathing glial cells from adult rat olfactory nerve, serving as a substrate for the regrowth of neurites from adult rat retinal ganglion cells. Immunocytochemically identified phenotypes present in primary cultures of olfactory nerve cells are described, and their ability to promote neurite outgrowth is compared with neonatal astrocytes and Schwann cells, with other nonglial cells, and with laminin. Ensheathing cell cultures were more effective than any other substrate tested and also directed the orientation of regrowing neurites. In comparison with cultured Schwann cells, which released neurotrophic factors into the culture medium, there was no evidence of a similar activity in ensheathing cell cultures. Combinations of ensheathing cell–conditioned medium and substrates of laminin, merosin, or 3T3 cells also failed to show the release of factors enhancing either survival or neurite outgrowth from retinal ganglion cells. Evidence is presented for a partial inhibition of neurite outgrowth in the presence of calcium channel antagonists or an intracellular calcium‐chelating reagent. This provides evidence for a contribution from an intracellular calcium signaling mechanism, possibly implicating ensheathing cell adhesion molecules in promoting neurite outgrowth. GLIA 25:256–269, 1999. © 1999 Wiley‐Liss, Inc.     

Lens injury stimulates adult mouse retinal ganglion cell axon regeneration via both macrophage- and lens-derived factors

In the present study the effects of lens injury on retinal ganglion cell axon/neurite re-growth were investigated in adult mice. In vivo, lens injury promoted successful regeneration of retinal ganglion cell axons past the optic nerve lesion site, concomitant with the invasion of macrophages into the eye and the presence of activated retinal astrocytes/Muller cells. In vitro, retinal ganglion cells from lens-lesioned mice grew significantly longer neurites than those from intact mice, which correlated with the presence of enhanced numbers of activated retinal astrocytes/Muller cells. Co-culture of retinal ganglion cells from intact mice with macrophage-rich lesioned lens/vitreous body led to increased neurite lengths compared with co-culture with macrophage-free intact lens/vitreous body, pointing to a neurotrophic effect of macrophages. Furthermore, retinal ganglion cells from mice that had no lens injury but had received intravitreal Zymosan injections to stimulate macrophage invasion into the eye grew significantly longer neurites compared with controls, as did retinal ganglion cells from intact mice co-cultured with macrophage-rich vitreous body from Zymosan-treated mice. The intact lens, but not the intact vitreous body, exerted a neurotrophic effect on retinal ganglion cell neurite outgrowth, suggesting that lens-derived neurotrophic factor(s) conspire with those derived from macrophages in lens injury-stimulated axon regeneration. Together, these results show that lens injury promotes retinal ganglion cell axon regeneration/neurite outgrowth in adult mice, an observation with important implications for axon regeneration studies in transgenic mouse models.     

Growth of embryonic retinal neurites elicited by contact with Schwann cell surfaces is blocked by antibodies to L1

Explants from embryonic rat retina plated on Schwann cell monolayers were used to examine the mechanisms by which these central neurons interact with Schwann cell surfaces. Embryonic retinal explants extend neurites reliably on Schwann cell surfaces (Kleitman et al., 1988, J. Neurosci. 8: 653). Antibodies to molecules thought to be present on Schwann cell surfaces (laminin and the 217C antigen), on retinal neurite surfaces (Thy-1.1), or on both surfaces (L1) were tested for their ability to influence this neurite growth. Of these, only antibodies to L1 were effective in blocking retinal neurite extension on Schwann cells. Inhibition of neurite growth by anti-L1 was shown to be specific to growth on Schwann cell surfaces because neurite growth on air-dried collagen (a substratum known to support retinal neurite outgrowth) was not affected. This blockage was dose-dependent. At a low titer of anti-L1 Fab fragments defasciculation of neurites was prominent; at high titers 95% of neurite outgrowth was inhibited. This virtual elimination of the ability of Schwann cell surfaces to support embryonic retinal neurite growth in the presence of antibodies to L1 indicates that binding of the L1 molecule is a critical component of the mechanism by which Schwann cells foster the growth of these neurites. The present experiments concur with the growing body of evidence that L1 plays an important role in supporting neurite growth on cell surfaces and raise the possibility that L1 may also mediate the striking ability of adult retinal axons to regenerate in a peripheral nerve environment.     

Membrane-bound CSPG mediates growth cone outgrowth and substrate specificity by Schwann cell contact with the DRG neuron cell body and not via growth cone contact

The central nervous system and peripheral nervous system (CNS/PNS) contain factors that inhibit axon regeneration, including myelin-associated glycoprotein (MAG), the Nogo protein, and chondroitin sulfate proteoglycan (CSPG). They also contain factors that promote axon regeneration, such as nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF). Axon regeneration into and within the CNS fails because the balance of factor favors inhibiting regeneration, while in the PNS, the balance of factor favors promoting regeneration. The balance of influences in the CNS can be shifted toward promoting axon regeneration by eliminating the regeneration-inhibiting factors, overwhelming them with regeneration-promoting factors, or making axon growth cones non-receptive to regeneration-inhibiting factors. The present in vitro experiments, using adult rat dorsal root ganglion (DRG) neurons, were designed to determine whether the regeneration-inhibiting influences of Schwann cell CSPG are mediated via Schwann cell membrane contact with the DRG neuron cell body or their growth cones. The average longest neurite of neurons in cell body contact with Schwann cells was 7.4-fold shorter than those of neurons without Schwann cell-neuron cell body contact (naked neurons), and the neurites showed substrate specificity, growing only on the Schwann cell membranes and not extending onto the laminin substrate. The neurites of naked neurons showed no substrate specificity and extended over the laminin substrate, as well as onto and off the Schwann cells. After digesting the Schwann cell CSPG with the enzyme C-ABC, neurons in cell body contact with Schwann cells extended neurites the same length as those of naked neurons, and their neurites showed no substrate selectivity. Further, the neurites of naked neurons were not longer than those of naked neurons not exposed to C-ABC. These data indicate that the extent of neurite outgrowth from adult rat DRG neurons and substrate specificity of their growth cone is mediated via contact between the Schwann cell membrane-bound CSPG and the DRG neuron cell body and not with their growth cones. Further, there was no apparent influence of diffusible or substrate-bound CSPG on neurite outgrowth. These results show that eliminating the CSPG of Schwann cells in contact with the cell body of DRG neurons eliminates the sensitivity of their growth cones to the CSPG-induced outgrowth inhibition. This may in turn allow the axons of these neurons to regenerate through the dorsal roots and into the spinal cord.     

Embryonic Optic Nerve Tissue Fails to Support Neurite Outgrowth by Central and Peripheral Neurons In Vitro

The failure of axon regeneration in the injured mammalian central nervous system has been ascribed, in part, to the inhibitory effects of myelin proteins. To investigate the influence of myelination on neurite growth and regeneration by both central nervous system and peripheral nervous system neurons, isolated rat neonatal retinal ganglion cells and adult and neonatal dorsal root ganglion neurons were cultured on cryostat sections of both immature unmyelinated and mature fully myelinated adult rat optic nerve. In agreement with earlier studies using neonatal peripheral neurons, the adult optic nerve failed to support neurite outgrowth from any of the neurons tested. A new finding was that tissue sections from unmyelinated optic nerve (aged embryonic days 18 and 20, and postnatal days 1–3), also failed to support the growth of neurites from neonatal retinal ganglion cells and both neonatal and adult dorsal root ganglion neurons. Neonatal retinal ganglion cells also failed to extend neurites on sections of pre-degenerated sciatic nerve, a tissue shown in our previous work to be a good substratum for supporting neurite growth for both neonatal and adult DRG neurons. These results suggest that cells in the immature optic nerve either express widely acting axon growth inhibitory molecules unrelated to previously described myelin proteins, or do not synthesize appropriate axon growth promoting molecules. They also reveal that, for axon regeneration, central nervous system and peripheral sensory neurons require distinct substratum interactions.     

Adult olfactory ensheathing glia promote the long-distance growth of adult retinal ganglion cell neurites in vitro

In vivo, transplanted adult olfactory ensheathing glia (OEG) and adult Schwann cells (SC) can support the regrowth of at least some transected axons within adult CNS neuropil. In the present study, we developed an in vitro adult rat retinal explant model to explore the influence of primary adult SC and OEG on retinal ganglion cell (RGC) neurite regrowth in the presence of glial cells endogenous to the retina. Retinal quadrants were plated RGC-side down onto aclar hats coated with either pure collagen (type 1), collagen with OEG, collagen with SCs, or collagen coated with both OEG and SCs. Regrowing retinal neurites extended onto the pure collagen substrate, largely in association with astrocytes that migrated out from the explants (mean number of neurites: 144+/-65 SEM). The additional presence of OEG (669+/-122), but not SCs (97+/-41), supported the regrowth of significantly greater numbers of RGC neurites. Furthermore, this OEG-stimulated regeneration was over significantly greater distances; >68% of neurites extended >500 microm from the explant, compared with explants plated onto SCs or collagen alone (15% and 29%, respectively). When OEG and SCs were co-cultured the number of regenerating neurites was reduced (397+/-81) compared with the pure OEG treatment. Analysis of explants on pure collagen substrates fed with media conditioned by purified OEG or SC showed no increase in neurite outgrowth compared with control treatments, suggesting that the enhanced growth in the presence of OEG is a contact-mediated effect. The observed differences between the abilities of OEG and SC to support the growth of CNS-derived fibers in the presence of astrocytes support the suggestion that OEG may be better suited for direct transplantation into CNS neuropil following injury.     

Neurite-promoting factors and extracellular matrix components accumulating in vivo within nerve regeneration chambers

The outgrowth of neurites from cultured neurons can be induced by the extracellular matrix glycoproteins, fibronectin and laminin, and by polyornithine-binding neurite-promoting factors (NPFs) derived from culture media conditioned by Schwann, or other cultured cells. We have examined the occurrence of fibronectin, laminin and NPFs during peripheral nerve regeneration in vivo. A previously established model of peripheral nerve regeneration was used in which a transected rat sciatic nerve regenerates through a silicone chamber bridging a 10 mm interstump gap. The distribution of fibronectin and laminin during regeneration was assessed by indirect immunofluorescence. Seven days after nerve transection the regenerating structure within the chamber consisted primarily of a fibrous matrix which stained with anti-fibronectin but not anti-laminin. At 14 days, cellular outgrowths from the proximal and distal stumps (along which neurites grow) had entered the fibronectin-containing matrix, consistent with a role of fibronectin in promoting cell migration. Within these outgrowths non-vascular as well as vascular cell stained with anti-fibronectin and anti-laminin. Wihtin the degenerated distal nerve segment, cells characteristics of Bungner bands (rows of Schwann cells along which regenerating neurites extend) stained with anti-fibronectin and laminin. The fluid surrounding the regenerating nerve was found to contain NPF activity for cultured ciliary ganglia neurons which markedly increased during the period of neurite growth into the chamber. In previous studies using this particular neurite-promoting assay, laminin but to a much lesser extent fibronectin also promoted neurite outgrowth. Affinity-purified anti-laminin antibody failed to block chamber fluid NPF activity while completely blocking the neurite-promoting activity of laminin. These two results suggested that chamber fluid NPF activity did not consist of individual molecules of either fibronectin or laminin. The spatial and temporal distribution of insoluble fibronectin and laminin and the temporal correlation between chamber fluid NPF accumulation and neurite outgrowth support the possibility that these agents influence regenerative events including axonal elongation in vivo.     

Extracellular matrix from human umbilical cord-derived mesenchymal stem cells as a scaffold for peripheral nerve regeneration

The extracellular matrix, which includes collagens, laminin, or fibronectin, plays an important role in peripheral nerve regeneration. Recently, a Schwann cell-derived extracellular matrix with classical biomaterial was used to mimic the neural niche. However, extensive clinical use of Schwann cells remains limited because of the limited origin, loss of an autologous nerve, and extended in vitro culture times. In the present study, human umbilical cord-derived mesenchymal stem cells (hUCMSCs), which are easily accessible and more proliferative than Schwann cells, were used to prepare an extracellular matrix. We identiifed the morphology and function of hUCMSCs and investi-gated their effect on peripheral nerve regeneration. Compared with a non-coated dish tissue culture, the hUCMSC-derived extracellular matrix enhanced Schwann cell proliferation, upregulated gene and protein expression levels of brain-derived neurotrophic factor, glial cell-derived neurotrophic factor, and vascular endothelial growth factor in Schwann cells, and enhanced neurite outgrowth from dorsal root ganglion neurons. These ifndings suggest that the hUCMSC-derived extracellular matrix promotes peripheral nerve repair and can be used as a basis for the rational design of engineered neural niches.     

Extracellular matrix derived from astrocytes stimulates neuritic outgrowth from PC12 cells in vitro

The ability of astrocyte extracellular matrix to stimulate axonal elongation was examined using an in vitro model system. Extracellular matrix (ECM) was derived from primary cultures of rat astrocytes or meningeal cells, or from a cell line of bovine aortic endothelial cells. The cells were grown in 35-mm tissue culture dishes for 24 h and then removed non-enzymatically, leaving ECM attached to the surface of the culture dishes. Subsequently, PC12 pheochromocytoma cells were seeded onto the ECM and de novo neurite outgrowth was measured. Within 24 h, the PC12 cells exhibited profuse neuritic outgrowth on ECM derived from astrocytes and endothelial cells, without addition of exogenous nerve growth factor. Over a period of 4 days, the neurites continued to elongate and branched extensively. Little or no neuritic outgrowth was observed from PC12 cells grown on uncoated culture dishes or on culture dishes treated with astrocyte-conditioned medium. Only a slight stimulation of neurite outgrowth was observed on meningeal cell-derived ECM. These results indicate that astrocyte ECM, as well as endothelial cell ECM, possesses one or more molecular factors that can stimulate and maintain de novo axonal elongation from PC12 cells. It is suggested that immature astrocytes secrete neurite-promoting factors as a component of the ECM which act to stimulate and possibly guide the growth of axons during in vivo development.     

Optic nerve regeneration within artificial Schwann cell graft in the adult rat

We investigate whether an artificial graft made by cultured Schwann cell, extracellular matrix (ECM) and trophic factors can provide the environment for the regeneration of retinal ganglion cell (RGC) axons in adult rats. Six kinds of artificial grafts were used: ECM (control); ECM and Schwann cells; ECM, Schwann cells and either nerve growth factor, brain-derived neurotrophic factor (BDNF) and neurotrophin-4 (NT-4); ECM, Schwann cells, BDNF and NT-4, combined with intravitreal injection of BDNF. The grafts were transplanted onto the transected optic nerve. RGC regeneration was evaluated by dil retrograde labeling, immunohistochemistry, and electron microscopy at 3 weeks post-operation. The degree of dil labeled RGC was approximately 2% for ECM alone, and 10% for ECM and Schwann cells (p < 0.01). The labeling increased to approximately 20% by administration of neurotrophins. The addition of intravitreous BDNF injection resulted in highest labeling percentage of 30%. Immunohistochemical study showed that axons were association with GAP-43 and cell adhesion molecules. Neurotrophin receptors (Trk-A and Trk-B) were detected in nerve fibers both in the retina and in the graft. Remyelination was seen by electron microscopic observation. These results demonstrate that the regeneration of RGC axons is induced with the use of cultured Schwann cells and ECM as promoting factors for regrowth. The degree of regeneration was significantly increased by neurotrophins in the grafts and in the vitreous.     

Neurite guidance by non-neuronal cells in culture: preferential outgrowth of peripheral neurites on glial as compared to nonglial cell surfaces

Growing axons in the peripheral nervous system (PNS) encounter a variety of cellular and extracellular substrates. Since it is difficult to sort out the possible contributions of these diverse components of the extracellular environment to axonal guidance in vivo, I have developed an in vitro system to study neurite outgrowth on two classes of cells which may provide as substrates for growing axons during development or regeneration: glial cells, e.g., astrocytes and Schwann cells, and nonglial cells, e.g., fibroblasts. Although neurites from sympathetic and spinal sensory ganglia explants grew onto preformed monolayers of both glial and nonglial cells, glial cells were a markedly better substrate. On the glial cells the neurites extended at a rate of 25 to 30 micron/hr and traveled singly or in fine fascicles; their growth cones displayed long filopodia and migrated on the upper surface of the monolayer cells. Conditioned media experiments suggested that neurite outgrowth on glial cell monolayers was not mediated by soluble secreted factors. These results indicate that the glial cell surface is an attractive substrate for neurite outgrowth. In contrast, on nonglial cells the rate of outgrowth was only 10 to 15 micron/hr, large neurite fascicles were common, and the growth cones migrated beneath the monolayer cells in contact with the underlying artificial substrate. This location of the growth cone, coupled with the observation that conditioned medium from these cells promoted neurite outgrowth only when bound to artificial substrates, suggests that secreted substrate-associated components may be an important determinant of neurite outgrowth on nonglial cell monolayers.(ABSTRACT TRUNCATED AT 250 WORDS)     

Regeneration of axons from adult human retina in vitro

In an effort to establish an in vitro model of regenerating adult human central nervous system (CNS) neurons, we have investigated the potential for neurite growth from explants prepared from normal adult human retina. Eyes (donated for corneal transplantation) were removed within 2.0 h postmortem and stored on ice for 1.5 to 7.0 days. Retinal explants (1 mm2) were prepared and cultured at 37 degrees C on cellular or acellular substrata in an oxygen-rich, humidified atmosphere. Neurite outgrowth, visualized by neurofilament immunofluorescence, was observed only in the presence of Schwann cells, after a quiescent period of approximately 6 days in vitro. Of 50 explants cultured for 7 days or more on substantia containing Schwann cells, 43 showed evidence of viability in vitro and 28 extended neurites onto Schwann cell surfaces. Estimated rates of neurite growth on Schwann cell substrata reached a maximum of 0.22 mm/day. Neurites did not grow beyond the explant border onto culture substrata composed of either polylysine, laminin, type-I collagen, or monolayers of adult human retinal glia. These results demonstrate that under selected conditions, explants prepared from adult human retina harbor viable neurons and that Schwann cells promote and support regeneration of neurites from these neurons in vitro, allowing systematic analysis of conditions favorable to axonal regeneration from adult human CNS neurons.     

Transplanted Schwann cells, not olfactory ensheathing cells, myelinate optic nerve fibres

In a previous study we found that olfactory ensheathing cells transplanted into complete retrobulbar transections of the rat optic nerve mediated regeneration of severed retinal ganglion cell axons through the graft region. Although the regenerating axons were ensheathed by the transplanted cells, none of the regenerating axons became myelinated by either central or peripheral type myelin. In the present study we used the same operative procedure but transplanted Schwann cells instead of olfactory ensheathing cells. As with the olfactory ensheathing cell transplants the Schwann cells transplants also induced regeneration of the severed retinal ganglion cell axons into the graft region. In contrast to the situation with the olfactory ensheathing cell transplants, however, a considerable number of the regenerating axons became myelinated by peripheral type myelin produced by the transplanted Schwann cells. This observation identifies a further distinction between these two cell types which are phenotypically similar in many ways, but which have been shown to have major functional differences with regard to regeneration in spinal cord lesions.     

Retinal neurite growth on astrocytes is not modified by extracellular matrix, anti-L1 antibody, or oligodendrocytes.

Two factors that may influence the course of axonal regeneration in the central nervous system (CNS) are extracellular matrix (ECM) and cell surface molecules that may enhance or inhibit neurite outgrowth. Whereas cultured astrocytes have been reported to be a good substratum for neurite outgrowth, there is recent evidence that cultured oligodendrocytes are inhibitory. To test the influences of 1) ECM components, 2) the L1 adhesion molecule, and 3) the inhibitory potential of mature oligodendrocytes in the astrocytic environment, we have utilized a culture system in which neurites from embryonic rat retina grow vigorously on astrocyte monolayers. The major ECM components were assembled in neonatal rat cortical astrocyte-retina co-cultures only when the medium contained serum. In electron microscopic studies of serum containing cultures, retinal neurites were seen to be related to astrocyte surfaces but rarely were found in contact with ECM; in serum-free medium the association between neurites and astrocytes was similar. In addition, the growth of neurites was vigorous whether ECM was present or absent. Presence of antibodies against the cell surface adhesion molecule L1 did not inhibit retinal neurite elongation on glial fibrillary acidic protein-positive astrocytes. When oligodendrocytes from adult rat spinal cord were combined with the astrocytes, retinal neurites grew as well on the mixed glial population as on astrocytes alone. Immunostaining for galactocerebroside showed many oligodendrocyte processes to be aligned in the direction of neurite growth, suggesting association between the two cell types. This association was verified by electron microscopy. Furthermore, retinal explants extended neurites among myelin basic protein-positive oligodendrocytes cultured without astrocytes. Thus, the astrocyte surface is a strong promoter of neurite growth from embryonic rat retina. This growth did not depend upon either ECM or the L1 adhesion molecule. Because neurites grew on astrocytes in the presence of mature oligodendrocytes or among oligodendrocytes alone, we conclude that oligodendrocytes do not inhibit neurite growth under certain conditions.     

Different factors promote axonal regeneration of adult rat retinal ganglion cells after lens injury and intravitreal peripheral nerve grafting

We have investigated the differential mediators of the neurotrophic effects of intravitreal peripheral nerve grafting and lens injury on adult rat retinal ganglion cells (RGC). Lens injury and intravitreal peripheral nerve grafting both stimulated RGC neurite growth in vitro and axon regeneration past the optic nerve lesion site in vivo concomitant with activation of retinal glia and invasion of macrophages into the eye. These observations, together with the results of coculture studies using a macrophage-free intact peripheral nerve segment, a macrophage-free intact lens, a macrophage-rich peripheral nerve segment, or a macrophage-rich injured lens in retinal cultures suggest that the stimulation of RGC axon regeneration by lens injury and intravitreal peripheral nerve grafting share a common macrophage-derived component overlain by distinct lens-derived and peripheral nerve-derived neurotrophic factors, respectively. RGC axon regeneration following lens injury and intravitreal peripheral nerve grafting was similar in vivo, correlating with similar retinal glia activation whereas, in vitro, the level of RGC neurite outgrowth was significantly higher following intravitreal peripheral nerve grafting compared with lens injury, concomitant with the presence of increased numbers of activated retinal glia. This suggests that in vivo RGC axon regeneration induced by lens injury and peripheral nerve grafting may be limited, in part, by factors derived from activated retinal glia.     

Neuronal age influences the response to neurite outgrowth inhibitory activity in the central and peripheral nervous systems.

Axonal regeneration is abortive in the central nervous system (CNS) of adult mammals, but readily occurs in the injured peripheral nervous system (PNS). Recent experiments indicate an important role for both intrinsic neuronal features and extrinsic substrate properties in determining the propensity for axonal regrowth. In particular, certain components of adult mammalian CNS myelin have been shown to exert a strong inhibitory influence on neurite outgrowth. To determine whether the potent neurite outgrowth inhibitory activity found in CNS myelin may also be present in PNS myelin and to study the influence of neuronal age on neurite outgrowth, we used a cryoculture assay in which dissociated rat dorsal root ganglion (DRG) neurons of different ages were challenged to extend neurites on fractionated myelin and cryostat sections from the PNS (sciatic nerve and myelin-free degenerated sciatic nerve) and CNS (optic nerve) of adult rats. The CNS environment of the optic nerve did not support E17 to P8 DRG neurite adhesion or outgrowth. E17 DRG neurons, unlike their older counterparts, however, were able to attach and extend neurites onto normal sciatic nerve and onto purified PNS myelin. In contrast, a vigorous neurite outgrowth response from all the ages tested was observed on the myelin-free degenerated sciatic nerve. These results indicate that PNS myelin is a potent inhibitor of neurite outgrowth and that DRG neuronal age plays an important role in determining the propensity for neurite outgrowth and regenerative response on inhibitory PNS and CNS substrata.     

Enhanced peripheral nerve regeneration through a poled bioresorbable poly(lactic-co-glycolic acid) guidance channel

In this study we investigated the effects of materials prepared with electrical poling on neurite outgrowth in vitro and nerve regeneration in vivo. Neuro-2a cells were seeded on poled and unpoled poly(lactic-co-glycolic) (PLGA) films and observed at time periods 24, 48 and 72 h post-seeding. The percentage of cells with neurites and the neurites per cell were quantified using light microscopy. At 48 and 72 h post-seeding, both the number of cells with neurites and the neurites per cell were significantly increased on the poled films compared to those on unpoled films. An established rat sciatic nerve model was used for in vivo studies to assess the effects of PLGA guides, poled for two different periods, on peripheral nerve regeneration. Guides were inserted in rats to bridge a 1.0 cm gap created in the right sciatic nerve. After four weeks, nerves regenerated through poled guides displayed a significant increase in conduction velocity and significantly increased numbers of axons across the guides, as compared to nerves regenerating through an unpoled guidance channel. Electrical poling was shown to promote neurite growth, axon regeneration and the conduction rate of the repaired nerve. We concluded that guides prepared with electrical poling enhance peripheral nerve regeneration.     

Growth of adult rat retinal ganglion cell neurites on astrocytes

Astrocytes, as well as Schwann cells (SC), can provide suitable substrata for embryonic neurites during development, but their abilities to support adult regenerating neurites have not been directly compared. The aim of the present study was to determine the ability of astrocytes to promote adult rat retinal ganglion cell (RGC) regeneration in vitro and to compare this to previously determined growth on the surface of Schwann cells. We prepared Type I astrocytes (Raff et al: J. Neurosci. 3:1289-1300, 1983) from perinatal rats. These were subcultured and maintained in either a serum-free medium for at least 2 weeks (stellate astrocytes with little immunoreactivity for laminin) or in serum containing medium for 7 to 10 days (flat and polygonal astrocytes with immunoreactivity for laminin). Stellate astrocytes might therefore represent mature astrocytes in vivo (Ard and Bunge: J. Neurosci. 8:2844-2858, 1988), while flat astrocytes might resemble immature brain astrocytes (Liesi et al: J. Cell Biol. 96:920-924, 1983). Adult RGC survival and axonal regrowth on these glia populations was compared to that observed on different SC populations, as previously reported (Baehr and Bunge: Exp. Neurol. 106:27-40, 1989). Both astrocyte populations (either flat or stellate astrocytes) did not enhance RGC survival. Stellate astrocytes were less effective in supporting RGC axon regeneration than flat astrocytes. When these date were compared to RGC survival and axon growth on SC (Baehr and Bunge: Exp. Neurol. 106:27-40, 1989) only "activated" mature SC populations were superior to astrocytes in enhancing RGC survival and neurite regrowth.(ABSTRACT TRUNCATED AT 250 WORDS)