Intrinsic and extrinsic inhibition of oligodendrocyte development by rat retina

Cell patterning in the vertebrate CNS reflects the combination of localized cell induction, migration and differentiation. A striking example of patterning is the myelination of visual system. In many species, retinal ganglion cell axons are myelinated in the optic nerve but are unmyelinated in the retina. Here, we confirm that rat and mouse retina lack oligodendrocytes and their precursors and identify multiple mechanisms that might contribute to their absence. Soluble cues from embryonic retina inhibit the induction of oligodendrocytes from neural stem cells and their differentiation from optic nerve precursors. This inhibition is mediated by retinal-derived BMPs. During development BMPs are expressed in the retina and addition of the BMP antagonist Noggin reversed retinal inhibition of oligodendrocyte development. The lack of retinal oligodendrocytes does not simply reflect expression of BMPs, since no oligodendrocytes or their precursors developed when embryonic retinal cells were grown in the presence of Noggin and/or inductive cues such as Shh and IGF-1. Similarly, injection of Noggin into the postnatal rat eye failed to induce oligodendrocyte differentiation. These data combined with the proposed inhibition of OPC migration by molecules selectively expressed at the nerve retina junction suggest that multiple mechanisms combine to suppress retinal myelination during development.     

The astrocytes in the retina and optic nerve head of mammals: A special glia for the ganglion cell axons

Summary The neuroglia in the retina and the intraocular portion of the optic nerve of the monkey and cat has been examined by light and electron microscopy. In the retina two types of macroglial cells can be distinguished: 1) Müller cells, and 2) astrocytes. The bipolar radial glial cells of Müller penetrate the entire thickness of the retina and their basal processes align in the nerve fibre layer to form septa that fasciculate the axons of the ganglion cells. In contrast to the Müller cells, the retinal astrocytes are not homogeneously distributed throughout the retina; their number correlates with the thickness of the nerve fibre layer. The processes of the astrocytes are confined to the ganglion cell layer and to the nerve fibre layer. In the latter, the astrocytic processes run parallel to and between the axons of a given nerve fibre bundle. According to cytological criteria, the retinal astrocytes are protoplasmic. In the intraocular portion of the optic nerve, however, the astrocytes are fibrous and their processes run perpendicular to the axon bundles of the prelaminar portion of the optic nerve. Thus, because of their intimate morphological relationship to axons of the nerve fibre layer and the intraocular portion of the optic nerve, the astrocytes in the eye of the monkey and the cat may be considered as a special glia for the axons of ganglion cells.     

Differentiation and migration of astrocyte precursor cells and astrocytes in human fetal retina: relevance to optic nerve coloboma.

The presence of astrocyte precursor cells (APCs) and time course and topography of astrocyte differentiation during development were investigated by triple-label immunohistochemistry with intact fetal and adult human retinas. Throughout retinal development and adulthood, expression of Pax2 was restricted to cells of the astrocytic lineage. Three distinct stages of astrocytic differentiation were identified during development: i) Pax2+/vimentin+/GFAP- APCs; ii) Pax2+/vimentin+/GFAP+ immature perinatal astrocytes; and iii) Pax2+/vimentin-/GFAP+ mature perinatal astrocytes. In adult, cells with the antigenic phenotype of mature perinatal astrocytes were restricted to a region surrounding the optic nerve head (ONH), whereas cells at a fourth stage of differentiation, adult astrocytes (Pax2-/vimentin-/GFAP+), were apparent throughout the vascularized retina. APC appearance was centered around the ONH and preceded the appearance of perinatal astrocytes. A cluster of Pax2+ somas was also present in a small region surrounding the ONH at the ventricular surface of the developing retina, which suggests the existence of two distinct sites of astrocytic differentiation. The coincidence in the location of APCs and perinatal astrocytes at the ventricular zone with that of optic nerve colobomas, together with the association of Pax2 gene mutations with this condition, suggests that coloboma formation may result from impaired astrocyte differentiation during development.     

Comparison of the glial investment of normal and regenerating fiber bundles in the optic nerve and optic tectum of the goldfish and the Crucian carp

Summary The architecture of normal and regenerating nerve fiber bundles in the optic nerve of the goldfish and the Crucian carp was compared to that of the axonal fascicles in the optic tectum of these teleost species with the use of ultrathin sections and freeze-fracture replicas. The fascicles in the optic nerve are clearly demarcated by astrocytic processes, in contrast to the fascicles in the tectum. No astrocytes could be identified in the tectum; in this region processes of astrocytes or of radial glial cells do not form channeling structures reminiscent of those in the optic nerve. Furthermore, tectal blood vessels lack complete investments of glial processes. It can be assumed that at least in lower vertebrates a framework of astrocytic processes might be important for growth of optic fibers over large distances, i.e., from the eye to the tectum, but may be dispensable in the target region itself.     

Peculiar and typical oligodendrocytes are involved in an uneven myelination pattern during the ontogeny of the lizard visual pathway

We studied the myelination of the visual pathway during the ontogeny of the lizard Gallotia galloti using immunohistochemical methods to stain the myelin basic protein (MBP) and proteolipid protein (PLP/DM20), and electron microscopy. The staining pattern for the PLP/DM20 and MBP overlapped during the lizard ontogeny and was first observed at E39 in cell bodies and fibers located in the temporal optic nerve, optic chiasm, middle optic tract, and in the stratum album centrale of the optic tectum (OT). The expression of these proteins extended to the nerve fiber layer (NFL) of the temporal retina and to the outer strata of the OT at E40. From hatching onwards, the labeling became stronger and extended to the entire visual pathway. Our ultrastructural data in postnatal and adult animals revealed the presence of both myelinated and unmyelinated retinal ganglion cell axons in all visual areas, with a tendency for the larger axons to show the thicker myelin sheaths. Moreover, two kinds of oligodendrocytes were described: peculiar oligodendrocytes displaying loose myelin sheaths were only observed in the NFL, whereas typical medium electron-dense oligodendrocytes displaying compact myelin sheaths were observed in the rest of the visual areas. The weakest expression of the PLP/DM20 in the NFL of the retina appears to be linked to the loose appearance of its myelin sheaths. We conclude that typical and peculiar oligodendrocytes are involved in an uneven myelination process, which follows a temporo-nasal and rostro-caudal gradient in the retina and ON, and a ventro-dorsal gradient in the OT.     

Activation of epidermal growth factor receptors directs astrocytes to organize in a network surrounding axons in the developing rat optic nerve

In postnatal developing optic nerves, astrocytes organize their processes in a cribriform network to group axons into bundles. In neonatal rat optic nerves in vivo, the active form of EGFR tyrosine kinase is abundantly present when the organization of astrocytes and axons is most actively occurring. Blocking activity of EGFR tyrosine kinase during the development of rat optic nerves in vivo inhibits astrocytes from extending fine processes to surround axons. In vitro, postnatal optic nerve astrocytes, stimulated by EGF, organize into cribriform structures which look remarkably like the in vivo structure of astrocytes in the optic nerve. In addition, when astrocytes are co-cultured with neonatal rat retinal explants in the presence of EGF, astrocytes that are adjacent to the retinal explants, re-organize to an astrocyte-free zone into which neurites grow out from the retinal tissue. We hypothesize that in the developing optic nerve, EGFR activity directs the formation of a histo-architectural structure of astrocytes which surrounds axons and provides a permissive environment for axon development.     

Macrophages can modify the nonpermissive nature of the adult mammalian central nervous system

Although astrocytic gliosis has been linked to failure of axonal regeneration in the adult mammalian CNS, its role is not fully established. We used an in vitro assay to investigate the role of reactive astrocytes and macrophages in influencing axonal growth in the lesioned adult rat optic nerve. Soon after optic nerve transection, the nonpermissive nature of the optic nerve is altered to a permissive state near the lesion. This may account for injury-induced axonal sprouting and may contribute to the failure of these sprouts to elongate beyond the site of the lesion in vivo. We provide evidence that this lesion-induced change in the axonal growth-promoting properties of the CNS near the lesion may be produced by mononuclear phagocytes. In addition, several months after optic nerve transection, the degenerated nerves, which consist mainly of astrocytes and lack myelin, i.e., astrocytic "scar" tissue, are a good substrate for neurite growth. Taken together, these results suggest that in this in vitro system, substantial inhibitory effects are not associated with regions of astrocytic gliosis and that the nonpermissive nature of the CNS white matter can be modified by macrophages.     

Microtubules and filaments in the axons and astrocytes of early postnatal rat optic nerves

Changes in the population of microtubules and filaments within the cytoplasm of maturing axons and astrocytes have been studied during the early postnatal development of rat optic nerves. At birth, all of the axons are unmyelinated; most have a diameter of 0.2–0.3 µ and contain 4–10 microtubules. Neurofilaments do not occur with any frequency until about 5 days postnatal when they appear as individual groups, each containing 4–12. Subsequently, the neurofilaments of each group disperse so that they become more evenly distributed in mature axons. Developing astrocytes show similar but rather more dramatic changes. Most astrocytic processes contain only microtubules at birth, but during maturation filaments begin to appear in increasing numbers while microtubules become less common. This process continues until, in the mature fibrous astrocytes, filaments pack the cytoplasm and microtubules are rare. These observations suggest that the filaments within axons and astrocytes may be formed by the breakdown of microtubules.     

Control of axonophilic migration of oligodendrocyte precursor cells by Eph-ephrin interaction

The migration of oligodendrocyte precursor cells (OPCs) is modulated by secreted molecules in their environment and by cell-cell and matrix-cell interactions. Here, we ask whether membrane-anchored guidance cues, such as the ephrin ligands and their Eph receptors, participate in the control of OPC migration in the optic nerve. We postulate that EphA and EphB receptors, which are expressed on axons of retinal ganglion cells, interact with ephrins on the surface of OPCs. We show the expression of ephrinA5, ephrinB2 and ephrinB3 in the migrating OPCs of the optic nerve as well as in the diencephalic sites from where they originate. In addition, we demonstrate that coated EphB2-Fc receptors, which are specific for ephrinB2/B3 ligands, induce dramatic changes in the contact and migratory properties of OPCs, indicating that axonal EphB receptors activate ephrinB signaling in OPCs.Based on these findings, we propose that OPCs are characterized by an ephrin code, and that Eph-ephrin interactions between axons and OPCs control the distribution of OPCs in the optic axonal tracts, and the progress and arrest of their migration.     

pERK1/2 Peripheral Recruitment and Filopodia Protrusion Augment Oligodendrocyte Progenitor Cell Migration: Combined Effects of PDGF-A and Fibronectin

Oligodendrocyte progenitor cell (OPC) migration is critical for effective myelination of the central nervous system. Not only during normal myelination but also during remyelination, the growth factors (GFs) and extracellular matrix (ECM) protein affect the OPC migration. Studies showed the altered levels of GFs and ECM in the demyelinating lesions. In our earlier studies, we have shown that the effect of platelet-derived growth factor alpha (PDGF-A) on OPC migration is dose- and time-dependent. In that we have shown that the physiological concentration (1 ng/ml) of PDGF-A was unable to induce OPC migration at transient exposure (30 min). However, the involvement of ECM in the regulation of PDGF-A mediated OPC migration was not clear. In the present study, we have used fibronectin (FN) as ECM. PDGF-A and FN have similar and overlapping intracellular signaling pathways including the extracellular regulated kinases 1 and 2 (ERK1/2). Here we demonstrate how physiological concentration of PDGF-A combines with FN to augment OPC migration in vitro. The present study is first of its kind to show the importance of the synergistic effects of PDGF-A and FN on peripheral recruitment of phosphorylated/activated ERK1/2 (pERK1/2), actin-pERK1/2 co-localization, and filopodia formation, which are essential for the enhanced OPC migration. These findings were further confirmed by ERK1/2 inhibition studies, using the pharmacological inhibitor U0126. An understanding of these complex interactions may lead to additional strategies for transplanting genetically modified OPCs to repair widespread demyelinated lesions.     

Characterization of ectopic myelinated nerve fibers in the retina of a cynomolgus monkey (Macaca fascicularis)

We report histopathology of retinal myelination discovered in a cynomolgus monkey. It consisted of a uniform population of spindle cells arranged in fascicles within the retina at the optic disk. The present case is remarkable in that there is a paucity of reports describing myelinated retinal nerve fibers in monkeys.     

A role for Noggin in the development of oligodendrocyte precursor cells

Oligodendrocyte precursor cells (OPCs) can be differentiated in culture into either oligodendrocytes or type-2 astrocytes (2As), depending on the culture conditions. Whereas oligodendrocyte development can occur in the absence of inducing signals, 2A development apparently cannot. Fetal calf serum (FCS) and bone morphogenetic proteins (BMPs) are powerful inducers of 2A development in culture, but there is no compelling evidence that OPCs develop into astrocytes in vivo. We show here that BMPs are made by glial cells in the developing rat optic nerve, raising the question of why 2As do not normally develop in the optic nerve. We demonstrate that the BMP antagonist Noggin is strongly expressed by both OPCs and type-1 astrocytes in the developing optic nerve. We also show that depletion of Noggin by a small interference RNA inhibits OPC proliferation and induces 2A differentiation in the presence of a low, non-2A-inducing concentration of FCS. By contrast, enforced expression of Noggin in OPCs blocks FCS-induced 2A differentiation. These findings suggest that BMPs in FCS are largely responsible for the 2A-inducing activity of FCS and that Noggin may normally inhibit the formation of 2As in the developing CNS.     

Spatiotemporal Gradient of Astrocyte Development in the Chick Optic Tectum: Evidence for Multiple Origins and Migratory Paths of Astrocytes

Astrocytes have been considered to be transformed from radial glial cells that appear at early stage of development and play a scaffold-role for neuronal cell migration. Recent studies indicate that neuroepithelial cells in the spinal cord also give rise to astrocytes. However, the mode of astroglial generation and migration in the ventricular neuroepithelium remains poorly understood. In this study, we have utilized immunohistochemical and retroviral lineage tracing methods to characterize the developmental profiles of astrocytes in the chick optic tectum, which develops from both the neural tube and invasion of optic tract. Chick vimentin and glial fibrillary acidic protein (GFAP) were found as single bands at molecular weights consistent with those reported for mammalian species. Differential developmental trends were observed for both proteins with relative vimentin levels decreasing and GFAP levels increasing with embryonic age. We observed two streams of tectal GFAP-labeled astrocytes originated from the tectal ventricle (intrinsic origin) and the optic tract (extrinsic origin). The extrinsic astrocytes arose from the ventral neuroepithelium of the third ventricle, dispersed bilaterally to the optic tract, and subsequently to the outer layer of optic tectum, indicating migration of astrocytes along retinal ganglion cell axons. On the other hand, the intrinsic astrocytes from the tectal ventricular neuroepithelium appeared first in the ventral part of the optic tectum, and then in the lateral and dorsal tectum. The intrinsic tectal astrocytes closely associated with fascicles of vimentin-labeled radial glial cells, indicating a presumptive radial migration of astrocytes. These results demonstrated that the optic tectum contains heterogeneous populations of astrocytes developed from the different origins and routes of migration.     

Filaments in astrocytes and axons of mice optic nerve. A freeze-etching study

Fixed and nonfixed tissues from optic nerves of 20-day-old mice were examined with the electron microscope using the freeze-etching method. In this study the filaments of fibrious astrocytes are compared with those of axons. Both the astrocytic perikaron and the processes show a characteristic aspect in view of the arrangement and density of filaments. The most reliable criterion to distinguish them from nonmyelinated axons is the presence of areas with packed filaments. Furthermore, we demonstrated morphometrically that the filaments of axons and astrocytes from prefixed specimens had a statistically significant (p less than 0.001) smaller diameter (9.5 +/- 0.3 nm) than those from nonprefixed ones (10.5 +/- 0.3 nm). The diameters of filaments in axons and astrocytes are identical in fixed as well as in nonfixed material. The fine structure of filaments displays in addition to a helical form also a certain periodicity.     

Systemic Simvastatin Rescues Retinal Ganglion Cells from Optic Nerve Injury Possibly through Suppression of Astroglial NF-κB Activation

Neuroinflammation is involved in the death of retinal ganglion cells (RGCs) after optic nerve injury. The purpose of this study was to determine whether systemic simvastatin can suppress neuroinflammation in the optic nerve and rescue RGCs after the optic nerve is crushed. Simvastatin or its vehicle was given through an osmotic minipump beginning one week prior to the crushing. Immunohistochemistry and real-time PCR were used to determine the degree of neuroinflammation on day 3 after the crushing. The density of RGCs was determined in Tuj-1 stained retinal flat mounts on day 7. The effect of simvastain on the TNF-α-induced NF-κB activation was determined in cultured optic nerve astrocytes. On day 3, CD68-positive cells, most likely microglia/macrophages, were accumulated at the crushed site. Phosphorylated NF-κB was detected in some astrocytes at the border of the lesion where the immunoreactivity to MCP-1 was intensified. There was an increase in the mRNA levels of the CD68 (11.4-fold), MCP-1 (22.6-fold), ET-1 (2.3-fold), GFAP (1.6-fold), TNF-α (7.0-fold), and iNOS (14.8-fold) genes on day 3. Systemic simvastatin significantly reduced these changes. The mean ± SD number of RGCs was 1816.3±232.6/mm² (n = 6) in the sham controls which was significantly reduced to 831.4±202.5/mm² (n = 9) on day 7 after the optic nerve was crushed. This reduction was significantly suppressed to 1169.2±201.3/mm² (P = 0.01, Scheffe; n = 9) after systemic simvastatin. Simvastatin (1.0 µM) significantly reduced the TNF-α-induced NF-κB activation in cultured optic nerve astrocytes. We conclude that systemic simvastatin can reduce the death of RGCs induced by crushing the optic nerve possibly by suppressing astroglial NF-κB activation.     

Fine structure of the retino-optic nerve junction in the chicken

The aim of this study is to investigate a fine structure of the retino-optic nerve junction in the chicken. We especially focused on the myelin sheaths and astrocytes in the intraocular optic nerve (ION) and its adjoining parts. A part of the axons of retinal nerve fiber layer (NFL) were myelinated. Ganglion cell axons were ensheathed by loose myelin in the NFL and by a compact one in the ION and optic nerve (ON). Myelin structure changed from loose type to a compact one within the very narrow NFL-ION junction. Loose myelin forming cells are dark type of oligodendrocytes in the retina. From the most peripheral ON to the choroidal part of ION, astrocytes contained abundant microtubules. The optic nerve around the lamina cribrosa is exposed to mechanical force during eye movement. It is suggested that these microtubules may perform the cytoskeletal function. Astrocytes in the retinal part of ION had longer processes filled with abundant gliofilaments. They may provide the mechanical support for the ganglion cell axons, which are exposed directly to intraocular pressure. Although astrocytes in the retinal level of ION extended their processes into the retina, their soma was never found in the retina.     

Expression of αB-Crystallin in the Peripapillary Glial Cells of the Developing Chick Retina

Peripapillary glial cells (PPGCs) are a peculiar macroglia in avian species, located in the central retina adjacent to the optic nerve head. PPGCs have a similar shape and orientation to Müller cells, which traverse the entire layer of the retina; however, there are differences in protein expression between the two cell types. In the present study, we first demonstrated that PPGCs expressed αB-crystallin, which is not expressed in Müller cells, during retinal development. αB-crystallin was first faintly expressed in PPGCs of the E5 retina, adjacent to the optic nerve head. Further, αB-crystallin was exclusively expressed in PPGCs up to E14. The shape of these cells was bipolar with vitread and ventricular processes. The vitread processes of αB-crystallin+ PPGCs became finer at E18. Double labeling analysis clearly demonstrated that only vimentin+ or GFAP+ astrocytes were located in the optic nerve head and were demarcated from the retina by αB-crystallin+ PPGCs. Furthermore, we determined that αB-crystallin+ PPGCs, with a number of processes, completely wrapped the optic nerve head and were densely located in the junction of the optic nerve head and the retina in a whole mount preparation and in vertical-sectioned retinae. The results of present study, together with reports that retinal astrocytes migrate from the optic nerve head, suggest that PPGCs prevent astrocytes from migrating into the retina in avian species.     

Astrocytes as gate-keepers in optic nerve regeneration--a mini-review

Animals that develop without extra-embryonic membranes (anamniotes--fish, amphibians) have impressive regenerative capacity, even to the extent of replacing entire limbs. In contrast, animals that develop within extra-embryonic membranes (amniotes--reptiles, birds, mammals) have limited capacity for regeneration as adults, particularly in the central nervous system (CNS). Much is known about the process of nerve development in fish and mammals and about regeneration after lesions in the CNS in fish and mammals. Because the retina of the eye and optic nerve are functionally part of the brain and are accessible in fish, frogs, and mice, optic nerve lesion and regeneration (ONR) has been extensively used as a model system for study of CNS nerve regeneration. When the optic nerve of a mouse is severed, the axons leading into the brain degenerate. Initially, the cut end of the axons on the proximal, eye-side of the injury sprout neurites which begin to grow into the lesion. Simultaneously, astrocytes of the optic nerve become activated to initiate wound repair as a first step in reestablishing the structural integrity of the optic nerve. This activation appears to initiate a cascade of molecular signals resulting in apoptotic cell death of the retinal ganglion cells axons of which make up the neural component of the optic nerve; regeneration fails and the injury is permanent. Evidence specifically implicating astrocytes comes from studies showing selective poisoning of astrocytes at the optic nerve lesion, along with activation of a gene whose product blocks apoptosis in retinal ganglion cells, creates conditions favorable to neurites sprouting from the cut proximal stump, growing through the lesion and into the distal portion of the injured nerve, eventually reaching appropriate targets in the brain. In anamniotes, astrocytes ostensibly present no such obstacle since optic nerve regeneration occurs without intervention; however, no systematic study of glial involvement has been done. In fish, vigorously growing neurites sprout from the cut axons and within a few days begin to re-enervate the brain. This review offers a new perspective on the role of glia, particularly astrocytes, as "gate-keepers;" i.e., as being permissive or inhibitory, by comparison between fish and mammals of glial function during ONR.     

Rat optic nerve oligodendrocytes develop in the absence of viable retinal ganglion cell axons.

Retinal ganglion cell axons and axonal electrical activity have been considered essential for migration, proliferation, and survival of oligodendrocyte lineage cells in the optic nerve. To define axonal requirements during oligodendrogenesis, the developmental appearance of oligodendrocyte progenitors and oligodendrocytes were compared between normal and transected optic nerves. In the absence of viable axons, oligodendrocyte precursors migrated along the length of the nerve and subsequently multiplied and differentiated into myelin basic protein-positive oligodendrocytes at similar densities and with similar temporal and spatial patterns as in control nerves. Since transected optic nerves failed to grow radially, the number of oligodendrocyte lineage cells was reduced compared with control nerves. However, the mitotic indices of progenitors and the percentage of oligodendrocytes undergoing programmed cell death were similar in control and transected optic nerves. Oligodendrocytes lacked their normal longitudinal orientation, developed fewer, shorter processes, and failed to form myelin in the transected nerves. These data indicate that normal densities of oligodendrocytes can develop in the absence of viable retinal ganglion axons, and support the possibility that axons assure their own myelination by regulating the number of myelin internodes formed by individual oligodendrocytes.     

Heterogeneity of astrocytic membranes in the optic nerve and spinal cord of the lizard,Anolis carolinensis

Summary The architecture of astrocytic membranes in the optic nerve and the spinal cord of the lizard, Anolis carolinensis, was investigated by use of the freeze-fracturing technique. Whereas astrocytes in mammals reveal so-called orthogonal arrays of particles (OAPs) in their membranes, astrocytes in lower vertebrates lack these structures. This study demonstrates for the first time OAPs in astrocytes from a submammalian species. They were found commonly in the optic nerve and less frequently in the spinal cord. However, the OAPs in astrocytes of spinal cord were confined to midtrunk levels; the astrocytes in the caudal spinal cord failed to reveal OAPs.Additionally, the ependymal cells around the central canal did not show any OAPs, either in the thoracic or in the caudal spinal cord. They were interconnected by gap and tight junctions, which were not intercalated with each other.The findings support our current working hypothesis according to which the presence and absence of OAPs in astrocytes may be correlated with regenerative incapability or capability of CNS-structures; i.e., whereas the thoracic spinal cord in Anolis carolinensis is known to be incapable of regeneration after injury, the caudal spinal cord is regenerative.