Rapid and protracted phases of retinal ganglion cell loss follow axotomy in the optic nerve of adult rats

To investigate the short-and long-term effects of axotomy on the survival of central nervous system (CNS) neurons in adult rats, retinal ganglion cells (RGCs) were labelled retrogradely with the persistent market diI and their axons interrupted in the optic nerve (ON) by intracranial crush 8 or 10 mm from the eye or in intraorbital cut 0.5 or 3 mm from the eye. Labelled RGCs were counted in flat-mounted retinas at intervals from 2 weeks to 20 months after axotomy. Two major patterns of RGC loss were observed: (1) an inital abrupt loss that was confined to the first 2 weeks after injury and was more severe when the ON was cut close to the eye; (2) a slower, persistent decline in RGC densities with one-half survival times that ranged from approximately 1 month after intraorbital ON cut to 6 months after intracranial ON crush. A small population of RGCs (approximately 5%) survived for as long as 20 months after intraorbital axotomy. The initial loss of axotomized RGCs presumably results from time-limited perturbations related to the position of the ON injury. A. persistent lack of terminal connectivity between RGCs and their targets in the brain may contribute to the subsequent, more protracted RGC loss, but the differences between intraorbital cut and intracranial crush suggest that additional mechanisms are involved. It is unclear whether the various injury-related processes set in motion in both the ON and the retina exert random effects on all RGCs or act preferentially on subpopulations of these neurons. © 1993 John Wiley & Sons, Inc.     

Osteopontin drives retinal ganglion cell resiliency in glaucomatous optic neuropathy

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Local control of neurofilament accumulation during radial growth of myelinating axons in vivo. Selective role of site-specific phosphorylation

The accumulation of neurofilaments required for postnatal radial growth of myelinated axons is controlled regionally along axons by oligodendroglia. Developmentally regulated processes previously suspected of modulating neurofilament number, including heavy neurofilament subunit (NFH) expression, attainment of mature neurofilament subunit stoichiometry, and expansion of interneurofilament spacing cannot be primary determinants of regional accumulation as we show each of these factors precede accumulation by days or weeks. Rather, we find that regional neurofilament accumulation is selectively associated with phosphorylation of a subset of Lys-Ser-Pro (KSP) motifs on heavy neurofilament subunits and medium-size neurofilament subunits (NFMs), rising >50-fold selectively in the expanding portions of optic axons. In mice deleted in NFH, substantial preservation of regional neurofilament accumulation was accompanied by increased levels of the same phosphorylated KSP epitope on NFM. Interruption of oligodendroglial signaling to axons in Shiverer mutant mice, which selectively inhibited this site-specific phosphorylation, reduced regional neurofilament accumulation without affecting other neurofilament properties or aspects of NFH phosphorylation. We conclude that phosphorylation of a specific KSP motif triggered by glia is a key aspect of the regulation of neurofilament number in axons during axonal radial growth.     

Slits contribute to the guidance of retinal ganglion cell axons in the mammalian optic tract

RGC axons extend in the optic tracts in a manner that correlates with the expression in the hypothalamus and epithalamus of a soluble factor inhibitory to RGC axon outgrowth. Additionally, although the RGC axons extend adjacent to the telencephalon, they do not normally grow into this tissue. Here, we show that slit1 and slit2, known chemorepellents for RGC axons expressed in specific regions of the diencephalon and telencephalon, help regulate optic tract development. In mice lacking slit1 and slit2, a subset of RGC axons extend into the telencephalon and grow along the pial surface but not more deeply into this tissue. Surprisingly, distinct guidance errors occur in the telencephalon of slit1 -/-; slit2 +/- and slit1/2 -/- embryos, suggesting that the precise level of Slits is critical for determining the path followed by individual axons. In mice lacking both slit1 and slit2, a subset of RGC axons also project aberrantly into the epithalamus, pineal and across the dorsal midline. However, many axons reach their primary target, the superior colliculus. This demonstrates that Slits play an important role in directing the guidance of post-crossing RGC axons within the optic tracts but are not required for target innervation.     

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.     

Remodeling of retinal ganglion cell dendrites in the absence of action potential activity

The dendrites of ganglion cells in the retina have an excess number of spines and branches that are normally lost during the first postnatal month of development. We investigated whether this dendritic remodeling can be prevented when the action potential activity of ganglion cells is abolished by chronic intraocular injections of tetrodotoxin (TTX) during the first 4 or 5 postnatal weeks in the cat. Dendritic tree morphologies of alpha and beta ganglion cells from TTX-treated, non-TTX-treated (contralateral eye), and normal control retinae were compared after intracellular filling with Lucifer yellow. Qualitative observations and quantitative measurements indicate that TTX treatment does not prevent the normally occurring loss of spines and dendritic branches. Indeed, the dendritic trees of both alpha and beta cells in TTX injected eyes actually have even fewer spines and branches than normal cells at equivalent ages. However, because the total dendritic lengths of these cells are also reduced after TTX blockade, spine density is indistinguishable from untreated animals at the same age. In addition, although dendritic field areas are not altered with treatment, the complexity of the dendritic trees is reduced. These observations suggest that dendritic remodeling can occur in the absence of ganglion cell action potential activity. Thus, the factors that influence the dendritic and axonal development of retinal ganglion cells must differ, because similar TTX treatment during the period of axonal remodeling does have profound effects on the final pattern of terminal arborizations.     

Mesenchymal stem cell therapy in retinal and optic nerve diseases: An update of clinical trials

Retinal and optic nerve diseases are degenerative ocular pathologies which lead to irreversible visual loss. Since the advanced therapies availability, cell-based therapies offer a new all-encompassing approach. Advances in the knowledge of neuroprotection, immunomodulation and regenerative properties of mesenchymal stem cells(MSCs) have been obtained by several preclinical studies of various neurodegenerative diseases. It has provided the opportunity to perform the translation of this knowledge to prospective treatment approaches for clinical practice. Since 2008, several first steps projecting new treatment approaches, have been taken regarding the use of cell therapy in patients with neurodegenerative pathologies of optic nerve and retina. Most of the clinical trials using MSCs are in Ⅰ/Ⅱ phase, recruiting patients or ongoing, and they have as main objective the safety assessment of MSCs using various routes of administration. However, it is important to recognize that, there is still a long way to go to reach clinical trials phase Ⅲ-Ⅳ. Hence, it is necessary to continue preclinical and clinical studies to improve this new therapeutic tool. This paper reviews the latest progress of MSCs in human clinical trials for retinal and optic nerve diseases.     

Gap-43 immunoreactivity and axon regeneration in retinal ganglion cells of the rat

Retinal ganglion cells (RGCs) in rats were retrogradely labeled with the fluorescent tracer Fluorogold (FG) and subjected to GAP-43 and c-JUN immunocytochemistry to identify those RGSs that are capable of regenerating an axon. After optic nerve section (ONS) and simultaneous application of FG to the nerve stump (group 1 experiments), GAP-43 immunoreactive RGCs (between 2 and 21 days after ONS) always represented a subfraction of both FG-labeled (i.e., surviving) RGCs and RGCs exhibiting c-JUN. GAP-43 immunoreactive RGCs represented 22% of RGCs normally present in rat retinae and 25% of surviving RGCs at 5 days after ONS but were reduced to 2% and 1%, which is 6% and 5% of survivors at 14 and 21 days, respectively. In animals that received a peripheral nerve (PN) graft after ONS (group 2 experiments), RGCs with regenerating axons were identified by FG application to the graft at 14 and 21 days. When examined at 21 and 28 days, all FG-labeled RGCs exhibited GAP-43 immunoreactivity, and FG/GAP-43-labeled RGCs were 3% and 2% of those resent in normal rat retinae. In relation to surviving. RGCs GAP-43 immunoreactive RGCs represented 10% at both time points. FG-/GAP-43 labeled RGCs also exhibited c-JUN, but c-JUN immunoreactive RGCs were at both time points at least twice as numerous a FG-/GAP-43-labeled RGCs. These data suggest that regenerating axons in PN grafts derive specifically from GAP-43 reexpressing RGCs. Appearance of GAP-43 immunoreactivity may therefore identify those RGCs that are capable of axonal regeneration or sprouting. 1994 John Wiley & Sons, Inc.     

Alterations in the morphology of ganglion cell dendrites in the adult rat retina after optic nerve transection and grafting of peripheral nerve segments

Summary Transected ganglion cell axons from the adult retina are capable of reinnervating their central targets by growing into transplanted peripheral nerve (PN) segments. Injury of the optic nerve causes various metabolic and morphological changes in the retinal ganglion cell (RGC) perikarya and in the dendrites. The present work examined the dendritic trees of those ganglion cells surviving axotomy and of those whose severed axons re-elongated in PN grafts to reach either the superior colliculus (SC), transplanted SC, or transplanted autologous thigh muscle. The elaboration of the dendritic trees was visualized by means of the strongly fluorescent carbocyanine dye DiI, which is taken up by axons and transported to the cell bodies and from there to the dendritic branches. Alternatively, retinofugal axons regrowing through PN grafts were anterogradely filled from the eye cup with rhodamine B-isothiocyanate. The transection of the optic nerve resulted in characteristic changes in the ganglion cell dendrites, particularly in the degeneration of most of the terminal and preterminal dendritic branches. This occurred within the first 1 to 2 weeks following axotomy. The different types of ganglion cells appear to vary in their sensitivity to axotomy, as reflected by a rapid degeneration of certain cell dendrites after severance of the optic nerve. The most vulnerable cells were those with small perikarya and small dendritic fields (type II), whereas larger cells with larger dendritic fields (type I and III) were slower to respond and less dramatically affected. Regrowth of the lesioned axons in peripheral nerve grafts and reconnection of the retina with various tissues did not result in a significant immediate recovery of ganglion cell dendrites, although it did prevent some axotomized cells from further progression toward posttraumatic cell death.     

Ipsilateral retinal projections into the tectum during regeneration of the optic nerve in the cichlid fish Haplochromis burtoni: A dil study in fixed tissue

Retinal projections were experimentally manipulated in a bony fish to reveal conditions under which considerably enlarged ipsilateral projections developed and persisted. Three experimental groups were studied: animals after unilateral enucleation, after unilateral nerve crush, and after enucleation and crush of the remaining optic nerve. At 29 days after unilateral enucleation alone, no enhanced ipsilateral projection had developed. After nerve crush, however, large numbers of retinal fibers regenerated into the ipsilateral tectum. Retrogradely filled, ipsilaterally projecting ganglion cells were distributed throughout the entire retina. After 15 days regenerating retinal fibers covered the entire ipsilateral tectum. At later stages the ipsilateral projection showed progressive reduction in coverage of the tectum. Combining enucleation with nerve crush led to an ipsilateral projection that covered the tectum at 28 days and later. In this experimental situation the development of an ipsilateral projection appears to be a two-step process: (1) Fibers are rerouted to the ipsilateral side at the diencephalon, and (2) ipsilateral fibers persist in the tectum only in the absence of a contralateral projection while they appear to be eliminated in the other cases. © 1992 John Wiley & Sons, Inc.     

Conditional disruption of beta 1 integrin in Schwann cells impedes interactions with axons.

In dystrophic mice, a model of merosin-deficient congenital muscular dystrophy, laminin-2 mutations produce peripheral nerve dysmyelination and render Schwann cells unable to sort bundles of axons. The laminin receptor and the mechanism through which dysmyelination and impaired sorting occur are unknown. We describe mice in which Schwann cell-specific disruption of beta1 integrin, a component of laminin receptors, causes a severe neuropathy with impaired radial sorting of axons. beta 1-null Schwann cells populate nerves, proliferate, and survive normally, but do not extend or maintain normal processes around axons. Interestingly, some Schwann cells surpass this problem to form normal myelin, possibly due to the presence of other laminin receptors such as dystroglycan and alpha 6 beta 4 integrin. These data suggest that beta 1 integrin links laminin in the basal lamina to the cytoskeleton in order for Schwann cells to ensheath axons, and alteration of this linkage contributes to the peripheral neuropathy of congenital muscular dystrophy.     

中华蜜蜂工蜂视叶胚后发育过程中的细胞凋亡

本研究通过形态解剖和原位末端标记法(TUNEL),对中华蜜蜂Apis cerana cerana视叶胚后发育过程中的细胞凋亡进行了研究,结果表明:视叶内的细胞程序性死亡开始出现在1龄幼虫末期,随后凋亡细胞数量逐渐增加;在视叶的胚后发育过程中,细胞凋亡经历了3个高峰期,即2龄幼虫、5龄幼虫和蛹发育的第2天;在视叶3个部分的发育中,视髓层中细胞凋亡的数量远远多于视小叶和视神经节层,而视神经节层最少,说明了细胞凋亡的数量和位置与各部分结构发育的时间以及神经投射有关。广泛的细胞凋亡是蜜蜂视叶发育过程中的一个显著特征。     

Spatio-temporal pattern of programmed cell death in the developing Drosophila optic lobe

A large number of cells die via programmed cell death during the normal development of the Drosophila optic lobe. In this study, we report the precise spatial and temporal pattern of cell death in this organ. Cell death in the developing optic lobe occurs in two distinct phases. The first phase extends from the start of metamorphosis to the mid-pupal stage. During this phase, a large number of cells die in the optic lobe as a whole, with a peak of cell death at an early pupal stage in the lamina and medulla cortices and the region of the T2/T3/C neurons, and a smaller number of dead cells observed in the lobula plate cortex. The second phase extends from the mid-pupal stage to eclosion. Throughout this period, a small number of dying cells can be observed, with a small peak at a late pupal stage. Most of the dying cells are neurons. During the first phase, dying cells are distributed in specific patterns in cortices. The lamina cortex contains two distinct clusters of dying cells; the medulla cortex, four clusters; the lobula plate cortex, one cluster; and the region of the T2/T3/C neurons, one cluster. Many of the clusters maintain their distinct positions in the optic lobe but others extend the region they cover during development. The presence of distinct clusters of dying cells at different phases suggests that distinct mechanisms control cell death during different stages of optic lobe development in Drosophila.     

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

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

Laminar restriction of retinal macrophagic response to optic nerve axotomy in the rat

Following optic nerve transection, most of the retinal ganglion cells die. Their debris is promptly cleared by phagocytic cells. It is currently not known to what extent peripherally derived macrophages contribute to this activity. Using antibodies OX42 and ED‐1, phagocytic cells were labeled in the retinas of optic nerve lesioned adult rats. To distinguish whether the cells were reactive microglial or macrophagic in origin, blood‐borne monocytes were labeled with fluorescent microspheres while in the systemic circulation. Macrophages invaded the retina, but only in the nerve fiber layer, sparing the ganglion cell and other layers. These macrophages engulfed only the axonal debris from dying ganglion cells, not their degenerating cell bodies. These results indicate that although peripherally derived monocytic cells are recruited into the retrogradely degenerating retina, their role in clearing debris is limited to the optic fiber layer. © 1999 John Wiley & Sons, Inc. J Neurobiol 40: 55–66, 1999