Schwann cells in the regenerating fish optic nerve: Evidence that CNS axons,not the glia,determine when myelin formation begins

Fish optic nerve fibres quickly regenerate after injury, but the onset of remyelination is delayed until they reach the brain. This recapitulates the timetable of CNS myelinogenesis during development in vertebrate animals generally, and we have used the regenerating fish optic nerve to obtain evidence that it is the axons, not the myelinating glial cells, that determine when myelin formation begins. In fish, the site of an optic nerve injury becomes remyelinated by ectopic Schwann cells of unknown origin. We allowed these cells to become established and then used them as reporters to indicate the time course of pro-myelin signalling during a further round of axonal outgrowth following a second upstream lesion. Unlike in the mammalian PNS, the ectopic Schwann cells failed to respond to axotomy and to the initial outgrowth of new optic axons. They only began to divide after the axons had reached the brain. Shortly afterwards, small numbers of Schwann cells began to leave the dividing pool and form myelin sheaths. More followed gradually, so that by 3 months remyelination was almost completed and few dividing cells were left. Moreover, remyelination occurred synchronously throughout the optic nerve, with the same time course in the pre-existing Schwann cells, the new ones that colonised the second injury, and the CNS oligodendrocytes elsewhere. The optic axons are the only common structures that could synchronise myelin formation in these disparate glial populations. The responses of the ectopic Schwann cells suggest that they are controlled by the regenerating optic axons in two consecutive steps. First, they begin to proliferate when the growing axons reach the brain. Second, they leave the cell cycle to differentiate individually at widely different times during the ensuing 2 months, during the critical period when the initial rough pattern of axon terminals in the optic tectum becomes refined into an accurate map. We suggest that each axon signals individually for myelin ensheathment once it completes this process.     

Reciprocal Modulation Between Microglia and Astrocyte in Reactive Gliosis Following the CNS Injury

Reactive gliosis, also known as glial scar formation, is an inflammatory response characterized by the proliferation of microglia and astrocytes as well as astrocytic hypertrophy following injury in the central nervous system (CNS). The glial scar forms a physical and molecular barrier to isolate the injured area from adjacent normal nervous tissue for re-establishing the integrity of the CNS. It prevents the further spread of cellular damage but represents an obstacle to regrowing axons. In this review, we integrated the current findings to elucidate the tightly reciprocal modulation between activated microglia and astrocytes in reactive gliosis and proposed that modification of cellular response to the injury or cellular reprogramming in the glial scar could lead advances in axon regeneration and functional recovery after the CNS injury.     

Evidence for Cooperative Selection of Axons for Myelination by Adjacent Oligodendrocytes in the Optic Nerve

The cellular mechanisms that regulate the topographic arrangement of myelin internodes along axons remain largely uncharacterized. Recent clonal analysis of oligodendrocyte morphologies in the mouse optic nerve revealed that adjacent oligodendrocytes frequently formed adjacent internodes on one or more axons in common, whereas oligodendrocytes in the optic nerve were never observed to myelinate the same axon more than once. By modelling the process of axonal selection at the single cell level, we demonstrate that internode length and primary process length constrain the capacity of oligodendrocytes to myelinate the same axon more than once. On the other hand, probabilistic analysis reveals that the observed juxtaposition of myelin internodes among common sets of axons by adjacent oligodendrocytes is highly unlikely to occur by chance. Our analysis may reveal a hitherto unknown level of communication between adjacent oligodendrocytes in the selection of axons for myelination. Together, our analyses provide novel insights into the mechanisms that define the spatial organization of myelin internodes within white matter at the single cell level.     

Early reduction of microglia activation by irradiation in a model of chronic glaucoma

Glaucoma is a neurodegenerative disease that results in the progressive decline and ultimate death of retinal ganglion cells (RGCs). While multiple risk factors are associated with glaucoma, the mechanisms leading to onset and progression of the disease remain unknown. Molecular analysis in various glaucoma models has revealed involvement of non-neuronal cell populations, including astrocytes, Mueller glia and microglia, at early stages of glaucoma. High-dose irradiation was reported to have a significant long-term protective effect in the DBA/2J (D2) mouse model of glaucoma, although the cellular and molecular basis for this effect remains unclear. In particular, the acute effects of irradiation on specific cell populations, including non-neuronal cells, in the D2 retina and nerve have not been assessed. Here we report that irradiation induces transient reduction in proliferating microglia within the optic nerve head and glial lamina within the first week post-irradiation. This was accompanied by reduced microglial activation, with no effect on astrocyte gliosis in those regions. At later stages we confirm that early high-dose irradiation of the mouse head results in improvement of axonal structural integrity and anterograde transport function, without reduction of intraocular pressure. Thus reduced microglial activation induced by irradiation at early stages is associated with reduced optic nerve and retinal neurodegeneration in the D2 mouse model of glaucoma.     

天冬酰胺内肽酶和胶质细胞在创伤性脑损伤中的激活

摘要 目的：创伤性脑损伤（traumatic brain injury, TBI）缺乏安全有效的治疗手段,亟须寻找新的干预靶点。天冬酰胺内肽酶 (asparaginyl endopeptidase, AEP)在免疫和神经系统疾病中起重要作用,本研究观察了小鼠TBI模型中AEP的激活和变化,探讨AEP对脑损伤和修复的意义。方法：控制性皮层撞击法在小鼠右脑半球制作TBI损伤,在造模后的不同时间点,测定受损脑组织内的乳酸含量和AEP的活性变化,免疫荧光化学染色观察TBI之后3天的胶质细胞活化,以及AEP在其中的表达。结果：TBI造成乳酸在受损脑组织内逐渐堆积,导致小胶质细胞和星形胶质细胞的反应性活化和增生,AEP的上调和激活出现在TBI的继发性脑损伤阶段,AEP在小胶质细胞和星形胶质细胞内均出现上调。结论：AEP有可能参与调控TBI引发的胶质细胞活化,在神经损伤和修复中发挥重要作用。     

Effects of Wnt5a overexpression in spinal cord injury

Accordingly to its known function in corticospinal tract (CST) developmental growth, previous reports have shown an inhibitory role of Wnt5a in CST regeneration after spinal cord injury (SCI). Interestingly, it has been subsequently demonstrated that Wnt5a also modulates the developmental growth of non-CST axons and that different Wnt5a receptors are expressed in neurons, oligodendrocytes, NG2+ glial precursors and reactive microglia/macrophages and astrocytes after SCI. However, the role of Wnt5a in the response of these cell types, in the regeneration of non-CST axons and in functional recovery after SCI is currently unknown. To evaluate this, rats were subjected to spinal cord contusion and injected with a lentiviral vector generated to overexpress Wnt5a. Histological analyses were performed in spinal cord sections processed for the visualization of myelin, oligodendrocytes, neurons, microglia/macrophages, astrocytes, NG2+ glial precursors and serotonergic axons. Motor and bladder function recovery were also assessed. Further advancing our knowledge on the role of Wnt5a in SCI, we found that, besides its previously reported functions, Wnt5a overexpression elicits a reduction on neuronal cell density, the accumulation of NG2+ glial precursors and the descending serotonergic innervation in the affected areas, along with impairment of motor and bladder function recovery after SCI.     

BDNF improves axon transportation and rescues visual function in a rodent model of acute elevation of intraocular pressure

Optic neuropathies lead to blindness; the common pathology is the degeneration of axons of the retinal ganglion cells. In this study, we used a rat model of retinal ischemia-reperfusion and a one-time intravitreal brain-derived neurotrophic factor(BDNF)injection; then we examined axon transportation function, continuity, physical presence of axons in different part of the optic nerve, and the expression level of proteins involved in axon transportation. We found that in the disease model, axon transportation was the most severely affected, followed by axon continuity, then the number of axons in the distal and proximal optic nerve. BDNF treatment relieved all reductions and significantly restored function. The molecular changes were more minor,probably due to massive gliosis of the optic nerve, so interpretation of protein expression data should be done with some caution.The process in this acute model resembles a fast-forward of changes in the chronic model of glaucoma. Therefore, impairment in axon transportation appears to be a common early process underlying different optic neuropathies. This research on effective intervention can be used to develop interventions for all optic neuropathies targeting axon transportation.     

Trying to understand axonal regeneration in the CNS of fish.

In contrast to the situation in mammals and birds, neurons in the central nervous system (CNS) of fish--such as the retinal ganglion cells--are capable of regenerating their axons and restoring vision. Special properties of the glial cells and the neurons of the fish visual pathway appear to contribute to the success of axonal regeneration. The fish oligodendrocytes lack the axon growth inhibiting molecules that interfere with axonal extension in mammals. Instead, fish optic nerve oligodendrocytes support--at least in vitro--axonal elongation of fish as well as that of rat retinal axons. Moreover, the fish retinal ganglion cells re-express upon injury a set of growth-associated cell surface molecules and equip the regenerating axons throughout their path and up into their target, the tectum opticum with these molecules. This may indicate that the injured fish ganglion cells reactivate the cellular machinery necessary for axonal regrowth and pathfinding. Furthermore, the target itself provides positional marker molecules even in adult fish. These marker molecules are required to guide the regenerating axons back to their retinotopic home territory within the tectum.     

Gliogenesis in rat optic nerve: astrocytes are generated in a single wave before oligodendrocytes

The time of origin for astrocytes in the rat optic nerve was investigated to determine whether this cell type is generated in two waves, a first wave which occurs before the formation of oligodendrocytes and a second wave which occurs after the peak period of oligodendrocyte formation. To answer this question, multiple injections of radioactive thymidine were administered to rats after the peak period of oligodendrocyte production in the optic nerve and the animals were sacrificed several weeks after the first injection. Thymidine-labeled cells in the optic nerve were identified with the electron microscope. Of the labeled cells, greater than 80% are oligodendrocytes, 4% are microglia, 2% are astrocytes, and the remainder are unclassifiable. The thymidine-labeled cells in the nerve were not immunostained for glial fibrillary acidic protein (GFAP), a marker characteristic of astrocytes. The number of thymidine-labeled glia generated after the second postnatal week is a small fraction of the total number of glia generated neonatally. No evidence exists for a second wave of astrocyte formation in the rat optic nerve as has been suggested in a study by Miller et al. (1985, Dev. Biol. 111, 35-41); rather, the vast majority of astrocytes are generated during the first 2 postnatal weeks and these data are in keeping with classical studies of gliogenesis. The question of whether astrocytes in the rat optic nerve arise directly from division of an undifferentiated, common progenitor cell or from a cell committed to the astrocyte lineage was addressed by combining thymidine autoradiography with GFAP immunocytochemistry. Rats were sacrificed 1 hr after an injection of thymidine and their nerves were processed for GFAP immunocytochemistry and autoradiography. During the first postnatal week, many thymidine-labeled cells are immunostained for GFAP. These observations demonstrate that cells committed to the astrocyte lineage divide neonatally and give rise to additional astrocytes.     

The glial design of a teleost optic nerve head supporting continuous growth.

This study demonstrates the peculiarities of the glial organization of the optic nerve head (ONH) of a fish, the tench (Tinca tinca), by using immunohistochemistry and electron microscopy. We employed antibodies specific for the macroglial cells: glutamine synthetase (GS), glial fibrillary acidic protein (GFAP), and S100. We also used the N518 antibody to label the new ganglion cells' axons, which are continuously added to the fish retina, and the anti-proliferating cell nuclear antigen (PCNA) antibody to specifically locate dividing cells. We demonstrate a specific regional adaptation of the GS-S100-positive Müller cells' vitreal processes around the optic disc, strongly labeled with the anti-GFAP antibody. In direct contact with these Müller cells' vitreal processes, there are S100-positive astrocytes and S100-negative cells ultrastructurally identified as microglial cells. Moreover, a population of PCNA-positive cells, characterized as glioblasts, forms the limit between the retina and the optic nerve in a region homologous to the Kuhnt intermediary tissue of mammals. Finally, in the intraocular portion of the optic nerve there are differentiating oligodendrocytes arranged in rows. Both the glioblasts and the rows of developing cells could serve as a pool of glial elements for the continuous growth of the visual system.     

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.     

A developmental and ultrastructural study of the optic chiasma in Xenopus

The structure of the optic chiasma in Xenopus tadpoles has been investigated by light and electron microscopy. Where the optic nerve approaches the chiasma, a tongue of cells protrudes from the periventricular cell mass into the dorsal part of the nerve. Glial processes from this tongue of cells ensheath fascicles of optic axons as they enter the brain. Coincident with this partitioning, the annular arrangement of axons in the optic nerve changes to the laminar organization of the optic tract. Beyond the site of this rearrangement, all newly growing axons accumulate in the ventral-most part of the nerve and pass into the region between the periventricular cells and pia which we have called the 'bridge'. This region is characterized by a loose meshwork of glial cell processes, intercellular spaces and the presence of both optic and nonoptic axons. In the bridge, putative growth cones of retinal ganglion cell axons are found in the intercellular spaces in contact with both the glia and with other axons. The newly growing axons from each eye cross in the bridge at the midline and pass into the superficial layers of the contralateral optic tracts. As the system continues to grow, previous generations of axon, which initially crossed in the existing bridge, are displaced dorsally and caudally, forming the deeper layers of the chiasma. At their point of crossing in the deeper layers, these fascicles of axons from each eye interweave in an intimate fashion. There is no glial segregation of the older axons as they interweave within the chiasma.     

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.     

A distinct effect of transient and sustained upregulation of cellular factor XIII in the goldfish retina and optic nerve on optic nerve regeneration

Unlike in mammals, fish retinal ganglion cells (RGCs) have a capacity to repair their axons even after optic nerve transection. In our previous study, we isolated a tissue type transglutaminase (TG) from axotomized goldfish retina. The levels of retinal TG (TG(R)) mRNA increased in RGCs 1-6weeks after nerve injury to promote optic nerve regeneration both in vitro and in vivo. In the present study, we screened other types of TG using specific FITC-labeled substrate peptides to elucidate the implications for optic nerve regeneration. This screening showed that the activity of only cellular coagulation factor XIII (cFXIII) was increased in goldfish optic nerves just after nerve injury. We therefore cloned a full-length cDNA clone of FXIII A subunit (FXIII-A) and studied temporal changes of FXIII-A expression in goldfish optic nerve and retina during regeneration. FXIII-A mRNA was initially detected at the crush site of the optic nerve 1h after injury; it was further observed in the optic nerve and achieved sustained long-term expression (1-40days after nerve injury). The cells producing FXIII-A were astrocytes/microglial cells in the optic nerve. By contrast, the expression of FXIII-A mRNA and protein was upregulated in RGCs for a shorter time (3-10days after nerve injury). Overexpression of FXIII-A in RGCs achieved by lipofection induced significant neurite outgrowth from unprimed retina, but not from primed retina with pretreatment of nerve injury. Addition of extracts of optic nerves with injury induced significant neurite outgrowth from primed retina, but not from unprimed retina without pretreatment of nerve injury. The transient increase of cFXIII in RGCs promotes neurite sprouting from injured RGCs, whereas the sustained increase of cFXIII in optic nerves facilitates neurite elongation from regrowing axons.     

Synantocytes: New functions for novel NG2 expressing glia

In the adult CNS, antibodies to the NG2 chondroitin sulphate proteoglycan (CSPG) label a large population of glia that have the antigenic phenotype of oligodendrocyte progenitor cells (OPC). However, NG2 expressing glia have the morphological phenotype of astrocytes, not OPC. We propose adult NG2 expressing glia are a distinct mature glial type, which we have called syantocytes or synantoglia after the Greek ‘to contact’, because they specifically contact neurons and axons at synapses and nodes of Ranvier, respectively. Synantocytes are highly complex cells that elaborate multiple branching processes and are an equally significant population in both white and grey matter. We provide evidence that phenotypically distinct synantocytes develop postnatally and that neither postnatal nor adult synantocytes depend on axons for their survival, indicating they respond with markedly different behaviours to the environmental cues and axonal signals that control the differentiation of OPC into oligodendrocytes. The primary response of synantocytes to changes in the CNS environment is a rapid and localised reactive gliosis. Reactive synantocytes interact intimately with astrocytes and macrophages at lesion sites, consistent with them playing a key role in the orchestration of scar formation that protects the underlying neural tissue. It is our hypothesis that synantocytes are specialised to monitor and respond to changes in the integrity of the CNS, by way of their cellular contacts, repertoire of plasmalemmal receptors and the NG2 molecule itself. To paraphrase Del Rio Hortega, we propose that synantocytes are the fifth element in the CNS, in addition to neurons, astrocytes, oligodendrocytes and microglia.     

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.     

Trying to understand axonal regeneration in the CNS of fish

In contrast to the situation in mammals and birds, neurons in the central nervous system (CNS) of fish—such as the retinal ganglion cells—are capable of regenerating their axons and restoring vision. Special properties of the glial cells and the neurons of the fish visual pathway appear to contribute to the success of axonal regeneration. The fish oligodendrocytes lack the axon growth inhibiting molecules that interfere with axonal extension in mammals. Instead, fish optic nerve oligodendrocytes support—at least in vitro—axonal elongation of fish as well as that of rat retinal axons. Moreover, the fish retinal ganglion cells re-express upon injury a set of growth associated cell surface molecules and equip the regenerating axons throughout their path and up into their target, the tectum opticum with these molecules. This may indicate that the injured fish ganglion cells reactivate the cellular machinery necessary for axonal regrowth and pathfinding. Furthermore, the target itself provides positional marker molecules even in adult fish. These marker molecules are required to guide the regenerating axons back to their retinotopic home territory within the tectum. © 1992 John Wiley & Sons, Inc.     

Accumulation of lipid inclusions in astrocytes of aging human optic nerve

We examined age-related changes in the human optic nerve (ON) from 10 postmortem donor eye samples (age: 21- to 94-year-old). In aged ON, many axons showed paucity of cytoskeleton, and possessed disorganized myelin that remained in the extracellular space. Lipid inclusions were detected in glia, as stained by oil red O, and these accumulated with aging. To identify and confirm which glial cell type possessed lipid inclusions, we performed immunohistochemistry (IHC) and transmission electron microscopy (TEM). Comparisons were made from TEM features and size of the glia immunolabeled with glial fibrillary acidic protein and glutamine synthetase (markers for astrocytes) and 2',3'-cyclic nucleotide 3'-phosphodiesterase (a marker for oligodendrocytes). It was found that lipid inclusions were restricted to the astrocytes having larger perikarya than the oligodendrocytes (IHC) and possessing filaments in cytoplasm (TEM). These astrocytes also possessed myelin debris and it is thus likely that those inclusions originated from degenerated myelin of the ON axons. These data indicate that astrocytes play a role in phagocytosis and clearance of disorganized myelin in aging human ON.