Mouse Optic Nerve Research Articles

Altered expression of CHL1 by glial cells in response to optic nerve injury and intravitreal application of fibroblast growth factor-2.

The close homologue of L1 (CHL1) is a member of the L1 family of cell recognition molecules. The protein is expressed by a variety of nerve cell types and subpopulations of glial cells in vivo and promotes elongation of neurites and survival of nerve cells in vitro. Here we demonstrate that glial cells up-regulate expression of CHL1 in response to an intraorbital crush of the adult mouse optic nerve. We also demonstrate that a single intravitreal application of fibroblast growth factor-2 (FGF-2) increases expression of CHL1 in retinal astrocytes and Müller cells. Elevated expression of CHL1 by glial cells in injured optic nerves and astrocytes and Müller cells in FGF-2-treated retinas suggests a role of the protein in the lesioned central nervous system. Results also suggest that trophic factors might exert part of their biological function by modifying expression of cell recognition molecules.

Coupling of calcium homeostasis to axonal sodium in axons of mouse optic nerve.

Axonal populations in neonatal and mature optic nerves were selectively stained with calcium dyes for analysis of calcium homeostasis and its possible coupling to axonal Na. Repetitive nerve stimulation causes a rise in axonal [Ca(2+)](i) the posttetanus recovery of which is impeded by increasing the number of action potentials in the tetanus. This effect is augmented in 4-aminopyridine (4-AP; 1 mM), which dramatically increases the calcium and presumably sodium load during the tetanus. Increasing axonal [Na](i) with the Na-ionophore monensin (4-50 microM) and ouabain (30 microM) retards posttetanus calcium decline, suggesting that efficient calcium clearance depends on a low level of axonal [Na](i). Posttetanus calcium clearance is not affected by K-mediated depolarization. To further examine coupling between axonal [Na](i) and [Ca(2+)](i), the resting axonal [Ca(2+)](i) was monitored as axonal [Na(+)](i) was elevated with ouabain, veratridine, and monensin. In all cases, elevation of axonal [Na(+)](i) evokes a calcium influx into axons. This influx is unrelated to activation of calcium channels but is consistent with calcium influx via reversal of the Na/Ca exchanger expected as a consequence of axonal [Na(+)](i) elevation. In conclusion, this study demonstrates that calcium homeostasis in the axons of the optic nerve is strongly coupled to axonal [Na(+)](i) in a manner consistent with the Na/Ca exchanger playing a major role in extruding calcium following nerve activity.

Development of astrocytes in the lamina cribrosa sclerae of the mouse optic nerve, with special reference to myelin formation.

In the mouse optic nerve, the optic nerve fiber layer in the retina, the optic papilla and the lamina cribrosa sclerae (LCS) just after penetrating the eyeball failed to generate myelin, whereas the optic nerve proper in the orbit was occupied by myelinated nerve fibers. The present study investigated development of the architecture of LCS, where the axons develop from unmyelinated to myelinated type, to elucidate how the initial part of axons was unmyelinated. At the LCS of the adult optic nerve, well developed astrocytes densely formed a cytoplasmic mesh-like frame through which unmyelinated fibers passed. The astrocytes here contained numerous and densely packed intermediate glial filaments and cell organelles. This framework formed by astrocytes appeared to be completed between 7 and 14 postnatal days before oligodendrocyte progenitors, migrated from the chiasm side, reached the proximal end of LCS, and began myelin formation. Thus the failure in myelin formation at the intraocular part and LCS possibly depended upon unsuccessful migration of oligodendrocytes beyond LCS constructed by specialized astrocytes, although other inhibitory factors for myelin formation, such as adhesion molecules distributed around LCS, may be unsolved.

Graded expression patterns of ephrin-As in the superior colliculus after lesion of the adult mouse optic nerve.

The idea has been put forward that molecules and mechanisms acting during development are re-used during regeneration in the adult, for example in response to traumatic injury in order to re-establish the functional integrity of neuronal circuits. Members of the Eph family of receptor tyrosine kinases and their 'ligands', the ephrins, play a prominent role during development of the retinocollicular projection in rodents, where EphA receptors and ephrin-As are expressed in gradients in both the retina and the superior colliculi (SC). We were interested in investigating whether EphA family members are also expressed or re-expressed in the adult after optic nerve lesion, since the presence of axon guidance information is an important prerequisite for a topographically appropriate re-connection by retinal ganglion cell (RGC) axons. This analysis was encouraged by results showing that RGC axons do not exert guidance preferences in response to membranes from adult unlesioned SC, but in response to membranes from the adult deafferented SC. We found a graded expression pattern of ephrin-As in the SC both before and after deafferentation, which was remarkably similar to those found during development. EphA receptor levels were reduced in the SC after deafferentation and the expression patterns of the EphB family were not changed. In particular, the presence of a graded ephrin-A expression in the deafferented SC suggests that - if robust regeneration of RGC axons can be achieved - topographic guidance information as a likely requirement for a functionally successful re-establishment of the retinocollicular projection is available.

Vaccination for Neuroprotection in the Mouse Optic Nerve: Implications for Optic Neuropathies

T-cell autoimmunity to myelin basic protein was recently shown to be neuroprotective in injured rat optic nerves. In the present study, using the mouse optic nerve, we examined whether active immunization rather than passive transfer of T-cells can be beneficial in protecting retinal ganglion cells (RGCs) from post-traumatic death. Before severe crush injury of the optic nerve, SJL/J and C3H.SW mice were actively immunized with encephalitogenic or nonencephalitogenic peptides of proteolipid protein (PLP) or myelin oligodendrocyte glycoprotein (MOG), respectively. At different times after the injury, the numbers of surviving RGCs in both strains immunized with the nonencephalitogenic peptides pPLP 190-209 or pMOG 1-22 were significantly higher than in injured controls treated with the non-self-antigen ovalbumin or with a peptide derived from beta-amyloid, a non-myelin-associated protein. Immunization with the encephalitogenic myelin peptide pPLP 139-151 was beneficial only when the disease it induced, experimental autoimmune encephalomyelitis, was mild. The results of this study show that survival of RGCs after axonal injury can be enhanced by vaccination with an appropriate self-antigen. Furthermore, the use of nonencephalitogenic myelin peptides for immunization apparently allows neuroprotection without incurring the risk of an autoimmune disease. Application of these findings might lead to a promising new approach for treating optic neuropathies such as glaucoma.

Phospho-dependent association of neurofilament proteins with kinesin in situ.

Recent studies demonstrate co-localization of kinesin with neurofilament (NF) subunits in culture and suggest that kinesin participates in NF subunit distribution. We sought to determine whether kinesin was also associated with NF subunits in situ. Axonal transport of NF subunits in mouse optic nerve was perturbed by the microtubule (MT)-depolymerizing drug vinblastine, indicating that NF transport was dependent upon MT dynamics. Kinesin co-precipitated during immunoprecipitation of NF subunits from optic nerve. The association of NFs and kinesin was regulated by NF phosphorylation, since (1) NF subunits bearing developmentally delayed phospho-epitopes did not co-purify in a microtubule motor preparation from CNS while less phosphorylated forms did; (2) subunits bearing these phospho-epitopes were selectively not co-precipitated with kinesin; and (3) phosphorylation under cell-free conditions diminished the association of NF subunits with kinesin. The nature and extent of this association was further examined by intravitreal injection of (35)S-methionine and monitoring NF subunit transport along optic axons. As previously described by several laboratories, the wave of NF subunits underwent a progressive broadening during continued transport. The front, but not the trail, of this broadening wave of NF subunits was co-precipitated with kinesin, indicating that (1) the fastest-moving NFs were associated with kinesin, and (2) that dissociation from kinesin may foster trailing of NF subunits during continued transport. These data suggest that kinesin participates in NF axonal transport either by directly translocating NFs and/or by linking NFs to transporting MTs. Both Triton-soluble as well as cytoskeleton-associated NF subunits were co-precipitated with kinesin; these data are considered in terms of the form(s) in which NF subunits undergo axonal transport.

Tenascin-R inhibits regrowth of optic fibers in vitro and persists in the optic nerve of mice after injury.

Tenascin-R, an extracellular matrix constituent expressed by oligodendrocytes and some neuronal cell types, may contribute to the inhibition of axonal regeneration in the adult central nervous system. Here we show that outgrowth of embryonic and adult retinal ganglion cell axons from mouse retinal explants is significantly reduced on homogeneous substrates of tenascin-R or a bacterially expressed tenascin-R fragment comprising the epidermal growth factor-like repeats (EGF-L). When both molecules are presented as a sharp substrate border, regrowing adult axons do not cross into the tenascin-R or EGF-L containing territory. All in vitro experiments were done in the presence of laminin, which strongly promotes growth of embryonic and adult retinal axons, suggesting that tenascin-R and EGF-L actively inhibit axonal growth. Contrary to the disappearance of tenascin-R from the regenerating optic nerve of salamanders (Becker et al., J Neurosci 19:813-827, 1999), the molecule remains present in the lesioned optic nerve of adult mice at levels similar to those in unlesioned control nerves for at least 63 days post-lesion (the latest time point investigated), as shown by immunoblot analysis and immunohistochemistry. In situ hybridization analysis revealed an increase in the number of cells expressing tenascin-R mRNA in the lesioned nerve. We conclude that, regardless of the developmental stage, growth of retinal ganglion cell axons is inhibited by tenascin-R and we suggest that the continued expression of the protein after an optic nerve crush may contribute to the failure of adult retinal ganglion cells to regenerate their axons in vivo.

Oligodendrocytes in brain and optic nerve express the beta3 subunit isoform of Na,K-ATPase.

The Na,K-ATPase, which catalyzes the active transport of Na(+) and K(+), has two principal subunits (alpha and beta) that have several genetically distinct isoforms. Most of these isoforms are expressed in the nervous system, but certain ones are preferentially expressed in glia and others in neurons. Of the beta isoforms, beta1 predominates in neurons and beta2 in astrocytes, although there are some exceptions. Here we demonstrate that beta3 is expressed in rat and mouse white matter oligodendrocytes. Immunofluorescence microscopy identified beta3 in oligodendrocytes of rat brain white matter in typical linear arrays of cell bodies between fascicles of axons. The intensity of stain peaked at 20 postnatal days. beta3 was identified in cortical oligodendrocytes grown in culture, where it was expressed in processes and colocalized with antibody to galactocerebroside. In the mouse and rat optic nerve, beta3 stain was seen in oligodendrocytes, where it colocalized with carbonic anhydrase II. For comparison, optic nerve was stained for the beta1 and beta2 subunits, showing distinct patterns of labelling of axons (beta1) and astrocytes (beta2). The C6 glioma cell line was also found to express the beta3 isoform preferentially. Since beta3 was not found at detectable levels in astrocytes, this suggests that C6 is closer to oligodendrocytes than astrocytes in the glial cell lineage.

Completion of myelin compaction, but not the attachment of oligodendroglial processes triggers K channel clustering

The characteristic localization of ion channels is crucial for the propagation of saltatory conduction in myelinated nerves. Voltage-gated Na+ channels are located at nodes of Ranvier while voltage-gated K+ channels are mainly found at juxtaparanodal regions. Recently, a humoral factor secreted by oligodendrocytes has been reported to induce clustering of Na+ channels in CNS axons. However, the molecular mechanisms for K+ channel clustering as well as the role of oligodendrocytes are still uncertain. To clarify whether myelin sheath itself can induce the distinct distribution of K+ channels, we have investigated the localization of K+ channels in adult and developing mouse optic nerves. The CNS axons from chronic demyelinating and hypomyelinating mice were also examined to determine if myelin sheaths were required for the maintenance of clusters. In all cases, the K+ channel clustering correlated well with compact myelin, but not with the presence of oligodendrocytes, suggesting that, in contrast to Na+ channel clustering, the formation of compact myelin is required for initiation as well as maintenance of K+ channel clustering. In addition, postsynaptic density protein-95 (PSD-95) or its highly related protein was found colocalized with K+ channels, suggesting that it may interact with K+ channels to form clusters at juxtaparanodal regions. J. Neurosci. Res. 58:752–764, 1999. © 1999 Wiley-Liss, Inc.

Completion of myelin compaction, but not the attachment of oligodendroglial processes triggers K+ channel clustering

The characteristic localization of ion channels is crucial for the propagation of saltatory conduction in myelinated nerves. Voltage-gated Na(+) channels are located at nodes of Ranvier while voltage-gated K(+) channels are mainly found at juxtaparanodal regions. Recently, a humoral factor secreted by oligodendrocytes has been reported to induce clustering of Na(+) channels in CNS axons. However, the molecular mechanisms for K(+) channel clustering as well as the role of oligodendrocytes are still uncertain. To clarify whether myelin sheath itself can induce the distinct distribution of K(+) channels, we have investigated the localization of K(+) channels in adult and developing mouse optic nerves. The CNS axons from chronic demyelinating and hypomyelinating mice were also examined to determine if myelin sheaths were required for the maintenance of clusters. In all cases, the K(+) channel clustering correlated well with compact myelin, but not with the presence of oligodendrocytes, suggesting that, in contrast to Na(+) channel clustering, the formation of compact myelin is required for initiation as well as maintenance of K(+) channel clustering. In addition, postsynaptic density protein-95 (PSD-95) or its highly related protein was found colocalized with K(+) channels, suggesting that it may interact with K(+) channels to form clusters at juxtaparanodal regions.

Matrix Metalloproteinase-9/Gelatinase B Is Required for Process Outgrowth by Oligodendrocytes

Oligodendrocytes (OLs) extend processes to contact axons as a prerequisite step in myelin formation. As the OL processes migrate toward their axonal targets, they modify adhesion to their substrate, an event that may be regulated by matrix metalloproteinases (MMPs). In the mouse optic nerve, MMP-9/gelatinase B increases during myelin formation. Although tissue inhibitor of metalloproteinase (TIMP)-3 also increases in parallel, the developing optic nerve has focally active MMPs demonstrable by in situ zymography. The distribution of proteolytic activity is similar to that of myelin basic protein, a marker of myelin formation. OLs in culture secrete MMP-9 and express active cell-associated metalloproteinases at the growing tips of their processes. TIMP-1 and a function-perturbing anti-MMP-9 antibody attenuate outgrowth of processes by OLs, indicating a requirement for MMP-9 in process outgrowth. Process reformation is retarded significantly in OLs cultured from MMP-9 null mice, as compared with controls, providing genetic evidence that MMP-9 is necessary for process outgrowth. These data show that MMP-9 facilitates process outgrowth by OLs in vivo and in culture.

Adenosine 5′ triphosphate evoked mobilization of intracellular calcium in central nervous system white matter of adult mouse optic nerve

Although it has been established that immature glial cells express functional purinergic receptors, the responsiveness of mature glial cells in vivo had not been elucidated. This question was addressed using fura-2 ratiometric measurements of [Ca2+]i in the adult mouse optic nerve, a central nervous system (CNS) white matter tract, taking advantage of the facts that (i), the optic nerve contains glial cells but not neurons and (ii), that fura-2 loads primarily astrocytes in isolated intact optic nerves. We show that adenosine 5′ triphosphate (ATP) evoked an increase in [Ca2+]i in a concentration-dependent manner with a half-maximal effect at 3 μm ATP, and with a rank order of agonist potency of ATP>ADP>α,β-methyline-ATP>UDP>adenosine. The results indicate mainly P2Y and P2X components, consistent with the in vitro astroglial purinergic receptor profile. The in vivo response of mature glia to ATP may be important in their response to CNS damage.

Thrombospondin expression in nerve regeneration II. comparison of optic nerve crush in the mouse and goldfish

Expression of the extracellular matrix molecule thrombospondin (TSP) was examined following retrobulbar crush injury of the goldfish and mouse optic nerve. TSP was present within the glia limitans and surrounding axon fascicles of the control normal goldfish optic nerve, but was absent from the normal mouse optic nerve. Following crush injury of the goldfish optic nerve, TSP expression increased dramatically along the path of regenerating axons and returned to near normal levels following axonal outgrowth. In contrast, during the unsuccessful attempt at regeneration following crush injury of the mouse optic nerve, TSP expression was present only in glial fibrillary acidic protein (GFAP)-negative, macrophage-rich regions distal to ganglion cell axons. These results indicate that TSP expression is increased in a temporal pattern along the path of regenerating goldfish optic nerve axons and therefore may be involved in successful central nervous system regeneration. The absence of TSP in the environment encountered by damaged mouse optic nerve axons may correlate with the lack of regeneration observed in the mouse optic nerve.

Maturational changes in cell surface antigen expression in the mouse retina and optic pathway

The distribution of the cell surface molecules M6 and L1 was studied using the immunohistochemistry and in situ hybridization in the developing and adult mouse retina and optic nerve. L1 is a cell adhesion molecule while M6 is a cell surface molecule homologous to the myelin protein proteolipid protein (PLP/DM20). Although both molecules were expressed in retina and optic nerves of embryonic and neonatal mice, our studies show that their patterns of postnatal expression are quite different. While L1 continues to be expressed in optic axons throughout adulthood, expression of M6 on optic axons declines after birth and instead becomes strongly expressed on Müller glial endfeet and in the inner plexiform layer. The modulation of these molecules after birth could provide clues to changing cell–cell interactions occurring in the proximal portion of the optic pathway.

Development of oligodendrocyte and myelination in mouse optic nerve

Development of oligodendrocyte and myelination in mouse optic nerve

Cellular retinaldehyde-binding protein is expressed by oligodendrocytes in optic nerve and brain

Cellular retinaldehyde-binding protein (CRALBP) is abundant in the retinal pigment epithelium and Müller glial cells of the retina, where it forms complexes with endogenous 11-cis-retinoids. We examined the distribution of CRALBP in extraretinal tissues using polyclonal antibodies (pAb) and monoclonal antibodies (mAb). A protein was detected by immunoblot analysis in extracts of bovine and rat brain and optic nerve but not in several other tissues. This protein had electrophoretic, chromatographic, and retinoid-binding properties identical to those of CRALBP from bovine retina. Comparison of the masses of tryptic peptides and of partial amino acid sequences derived from brain and retinal CRALBP indicated that the two proteins are probably identical. Immunoperoxidase cytochemistry and double labeling immunofluorescence revealed CRALBP(+) cells in brain that resembled oligondendrocytes and not astrocytes, microglial cells, or pinealocytes. In 11-day-old rat brain, approximately 11% of the CRALBP(+) cells were labeled with the Rip antibody, a marker for oligodendroglia. In developing rat optic nerve, the temporal appearance of CRALBP(+) cells corresponded to that of oligodendrocytes and not that of astrocytes. In adult rat and mouse optic nerves, the CRALBP(+) somata showed the same distribution as oligodendrocytes. No endogenous retinoids were associated with CRALBP isolated from dark-dissected adult bovine brain. The results suggest that CRALBP has functions in addition to retinoid metabolism and visual pigment regeneration.

Morphometric analysis of the postnatal mouse optic nerve following prenatal exposure to alcohol.

Pregnant female mice were divided on day 12 post coitum into a control and an experimental group. The experimental group was given a single intraperitoneal dose of 0.015 ml/g body weight of 25% solution of alcohol in distilled water while the control group was exposed to a similar weight related dose of normal saline. The optic nerves were isolated from the offspring of both control and experimental groups at wk 2, 3 and 5 (i.e. during the juvenile period of postnatal development) and analysed by light and electron microscopy. Although in both groups the optic nerve grew in size rapidly during the period studied, the rate of growth in the experimental groups lagged behind that of the controls. The difference was initially significant but tailed off, so that by wk 5 it was no longer significant. The time of initial onset and progression of myelinogenesis in the optic nerve of alcohol exposed mice also lagged behind that of controls. In both groups the size distribution of the myelinated nerve fibres in the optic nerve was unimodal with a positive skewing for all ages. The spectrum of size distribution of the nerve fibres was, however, broader in controls than in the corresponding experimental groups. With increasing age the proportion of small and medium size fibres was greater in the experimental group than in the controls, while for the large diameter fibres the reverse was observed. It is suggested that this study may shed light on the teratogenic effect of 'binge' drinking during pregnancy and that it is the critical period when exposure occurs that is more important than the duration of administration.

Changing course of growing axons in the optic chiasm of the mouse.

The distribution of retinofugal fibres has been studied by electron microscopy throughout the extent of the developing mouse optic nerve and chiasm at embryonic day (E) 16, in order to determine the course of fibre growth. Growth cones and mature axons, which are randomly distributed in bundles in the extracranial optic nerve, segregate in the juxtachiasmatic optic nerve. Here, growth cones accumulate in subpial regions amongst the endfeet of radial glia, whereas axons lie in the depths of the nerve. Surprisingly, however, growth cones move away from this region toward the ventricular zone in the lateral and midline parts of the chiasm, only to return to subpial regions once more before entering the optic tract, where fibres are again in an age-related order. Superficially, mature axons mingle with growth cones in the chiasm and near the beginning of the optic tract, suggesting that the age-related order begins to be reestablished before growth cones enter the tract. Deep and superficial regions of the pathway were examined in different planes of section. Specialised membrane relationships between retinofugal fibres and radial glial cells were also studied in deep and superficial regions of the lateral part of the chiasm. In addition, the distribution of retinofugal fibre bundles in the adult mouse was looked at by using light microscopy. The changing fibre positions noted in the embryo are maintained in the adult.

Myelinogenesis in the optic nerve of (C57BL x CBA) F1 hybrid mice: a morphometric analysis.

Myelinogenesis in the optic nerve of the mouse begins by the end of the first week of postnatal life and proceeds well into adulthood. The optic nerve from different postnatal age groups of (C57BL x CBA) F1 hybrid mice was analysed ultrastructurally and morphometrically. Features examined were the myelinated and unmyelinated nerve fibres, mean myelinated nerve fibre diameter, myelinated nerve fibre diameter spectrum, number of myelin sheath lamellae in relation to fibre diameter and age. Results obtained showed that the optic nerve of the mouse is populated entirely by unmyelinated nerve fibres at birth. Myelinogenesis begins at about the fifth postnatal day (pnd), with clearer evidence by the 6th pnd. It starts selectively with the largest fibres and then progresses to involve other fibres of decreasing diameter. After the onset, myelinogenesis progresses with increasing rapidity, becoming most intense from the second week up to the fifth week, so that by the end of the fifth week about 73% of fibres have become myelinated. From then onward, the process proceeds at a progressively diminishing rate until, by the 16th week, virtually all the remaining 27% fibres are myelinated. At onset, the myelin sheath is loosely wrapped around the axons, but with increasing age the myelin lamellae become more compact around the axons. The population of unmyelinated fibres continued to diminish so that at the completion of the process (16th week), they were only very rarely encountered. Myelinogenesis in the optic nerve of the mouse appears to follow a similar course to that seen in other central tracts having a similar fibre diameter spectrum.

Order – Disorder Phenomena in Myelinated Nerve Sheaths. VI.The Effects of quaking, jimpyand shivererMutations: An X-ray Scattering Study of Mouse Sciatic and Optic Nerves

We report the X-ray scattering study of sciatic and optic nerve myelin from shiverer, jimpyand quakingmice mutants and from the corresponding controls. These three mutations are known to affect dramatically central nervous system (CNS) myelin and to induce comparatively minor alterations in peripheral nervous system (PNS) myelin. Scattering experiments and data reduction were carried out using the techniques and algorithms developed in our laboratory and previously applied to several problems involving the structure of myelin. In sciatic nerve the fraction of myelin elementary pairs of membranes (total myelin) decreases in shivererand quakingnerves (by approximately 30%) but not in jimpynerves; in all three mutants the fraction of myelin membrane pairs that are not regularly stacked in the sheaths (loose myelin), the average number of membranes per sheath and the packing disorder are the same as in the control nerves; the repeat distance Dand the membrane distance D cytacross the cytoplasmic space increase in shivererand decrease in jimpy; in quaking, Dalso decreases and the decrease is smaller than in jimpyand is not specific for D cyt; small changes are also observed in the electron density profiles. As for the optic nerve the myelin content decreases dramatically in the three mutants; the very weak signal attests to a tiny amount of pairs of membranes structurally similar to normal CNS myelin. It is surprising that the structure of CNS myelin should be almost normal in the absence of the major structural components, namely myelin basic protein (MBP) for shivererof proteolipid protein (PLP) for jimpy. The question arises whether the composition of the residual pairs of membranes, operationally identified as myelin in the X-ray scattering experiments, mirrors the composition determined by chemical means on the fraction of nerve tissue histologically identified as myelin, or whether in all circumstances it remains approximately the same.