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.     

Myocilin expression in the astrocytes of the optic nerve head

We investigated the expression of myocilin in the optic nerve head of porcine eyes by Western blotting and immunohistochemical staining. Myocilin was localized in the nucleus, centrosome, glial filament, mitochondria, and some parts of the cell membranes of the astrocytes. Myocilin was also detected at the edge-feet portion of the processes of astrocytes adjacent to the inner limiting membrane and blood vessel wall. The astrocytes are the major cell population in the optic nerve head, contributing to the architecture of the nerve axon and blood vessels. Therefore, myocilin gene mutation and change of myocilin protein are likely to affect the architecture of the optic nerve head and induce various forms of glaucomatous optic nerve damage.     

Immuno-ultrastructural localization of sodium channels at nodes of Ranvier and perinodal astrocytes in rat optic nerve

Immuno-electron microscopic localization of sodium channels at nodes of Ranvier within adult optic nerve was demonstrated with polyclonal antibody 7493. The 7493 antisera, which is directed against purified sodium channels from rat brain, recognizes a 260 kDa protein in immunoblots of the crude glycoprotein fraction from adult rat optic nerve. Intense immunoreactivity with 7493 antisera was observed at nodes of Ranvier. Axon membrane at the node was densely stained, whereas paranodal and internodal axon membrane did not exhibit immunoreactivity. The axoplasm beneath the nodal membrane displayed variable immunostaining. Neither terminal paranodal oligodendroglial loops nor oligodendrocyte plasmalemma were immunoreactive with 7493 antisera. However, perinodal astrocyte processes exhibited intense immunoreactivity with the anti-sodium channel antisera. Optic nerves incubated with pre-immune sera, or with 7493 antisera that had been pre-adsorbed with purified sodium channel protein, displayed no immunoreactivity. These results demonstrate localization of sodium channels at high density at mammalian nodes of Ranvier and in some perinodal astrocyte processes. The latter observation offers support for an active role for perinodal astrocyte processes in the aggregation of sodium channels within the axon membrane at the node of Ranvier.     

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.     

Isolation of 10-nm filaments from astrocytes in the mouse optic nerve

Astroglial filaments approximately 10 nm in diameter were isolated from degenerated mouse optic nerves by Triton X-100 and DNase I treatments followed by sucrose density gradient centrifugation. 2-4 wk after bilateral enucleation, optic nerves contained virtually a single population of 10-nm filaments (astroglial filaments), free from neurofilaments. In negative-staining and thin-section electron microscopy, the isolated filaments were seen as nonbranching linear structures with smooth contour, and were morphologically identical to those in situ. Sodium dodecyl sulfate polyacrylamide gel electrophoresis revealed the isolated filaments to be composed of two major polypeptides with molecular weights of 45,000 and 55,000, present in an approximate molar ratio of 1:1. These findings, together with the results of one-dimensional peptide mapping and solubility study, indicate that the astroglial filaments in the mouse optic nerve are primarily composed of these two polypeptides.     

Platelet-activating factor stimulates sodium-hydrogen exchange in ventricular myocytes

Sodium-hydrogen exchanger (NHE), the principal sarcolemmal acid extruder in ventricular myocytes, is stimulated by a variety of autocrine/paracrine factors and contributes to myocardial injury and arrhythmias during ischemia-reperfusion. Platelet-activating factor (PAF; 1-o-alkyl-2-acetyl-sn-glycero-3-phosphocholine) is a potent proinflammatory phospholipid that is released in the heart in response to oxidative stress and promotes myocardial ischemia-reperfusion injury. PAF stimulates NHE in neutrophils and platelets, but its effect on cardiac NHE (NHE1) is unresolved. We utilized quiescent guinea pig ventricular myocytes bathed in bicarbonate-free solutions and epifluorescence to measure intracellular pH (pH(i)). Methylcarbamyl-PAF (C-PAF; 200 nM), a metabolically stable analog of PAF, significantly increased steady-state pH(i). The alkalosis was completely blocked by the NHE inhibitor, cariporide, and by sodium-free bathing solutions, indicating it was mediated by NHE activation. C-PAF also significantly increased the rate of acid extrusion induced by intracellular acidosis. The ability of C-PAF to increase steady-state pH(i) was completely blocked by the PAF receptor inhibitor WEB 2086 (10 μM), indicating the PAF receptor is required. A MEK inhibitor (PD98059; 25 μM) also completely blocked the rise in pH(i) induced by C-PAF, suggesting participation of the MAP kinase signaling cascade downstream of the PAF receptor. Inhibition of PKC with GF109203X (1 μM) and chelerythrine (2 μM) did not significantly affect the alkalosis induced by C-PAF. In summary, these results provide evidence that PAF stimulates cardiac NHE1, the effect occurs via the PAF receptor, and signal relay requires participation of the MAP kinase cascade.     

Proteomics analyses of human optic nerve head astrocytes following biomechanical strain

We investigate the role of glial cell activation in the human optic nerve caused by raised intraocular pressure, and their potential role in the development of glaucomatous optic neuropathy. To do this we present a proteomics study of the response of cultured, optic nerve head astrocytes to biomechanical strain, the magnitude and mode of strain based on previously published quantitative models. In this case, astrocytes were subjected to 3 and 12% stretches for either 2 h or 24 h. Proteomic methods included nano-liquid chromatography, tandem mass spectrometry, and iTRAQ labeling. Using controls for both stretch and time, a six-plex iTRAQ liquid chromatography- tandem MS (LC/MS/MS) experiment yielded 573 proteins discovered at a 95% confidence limit. The pathways included transforming growth factor β1, tumor necrosis factor, caspase 3, and tumor protein p53, which have all been implicated in the activation of astrocytes and are believed to play a role in the development of glaucomatous optic neuropathy. Confirmation of the iTRAQ analysis was performed by Western blotting of various proteins of interest including ANXA 4, GOLGA2, and αB-Crystallin.     

Imaging of rat optic nerve axons in vivo

In this protocol, we describe the imaging of single axons in the rat optic nerve in vivo. Axons are labeled through the intravitreal injection of adeno-associated viral vectors (AAVs) expressing a fluorophore (duration of the procedure ～1 h). Two weeks after intravitreal injection, the optic nerve is surgically exposed (duration ～1 h) and labeled axons are imaged with an epifluorescence microscope either for up to 8 h or repetitively on the following days. Additionally, intravitreal injection of calcium-sensitive dyes allows for imaging of intra-axonal calcium kinetics. This procedure enables the analysis of the morphological changes of degenerating axons in the optic nerve in different lesion paradigms, such as optic nerve crush, axotomy or pin lesion. Furthermore, the effects of pharmacological manipulations on axonal stability and axonal calcium kinetics in axons of the central nervous system can be studied in vivo.     

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.     

The differentiation of oligodendrocytes in the rat optic nerve

The time of appearance and the rate of accumulation of specific myelin lipids and proteins were measured in the rat optic nerve during the period from birth to 18 days. The appearance of the activities of several enzymes involved in the synthesis of these lipids was also monitored. Correlation of these biochemical data with previously known morphological findings indicated that the “active” oligodendrocytes (detected between 5 and 15 days after birth) displayed maximal levels of synthesis of the components of myelin, and that these cells appeared to be responsible for the initial synthesis of myelin. Both young and mature oligodendrocytes showed limited capacity to synthesize these compounds. Furthermore, induction of the enzymes involved in the synthesis of myelin components appeared to take place in a simultaneous, rather than a sequential manner.     

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.     

Electrical potentials from the eye and optic nerve of Strombus: effects of electrical stimulation of the optic nerve.

1. Photic stimulation of the mature eye of Strombus can evoke in the optic nerve 'on' activity in numerous small afferent fibres and repetitive 'off' bursts of afferent impulses in a smaller number of larger fibres. 2. Synchronous invasion of the eye by electrically evoked impulses in small optic nerve fibres (apparently the 'on' afferents, antidromically activated) can evoke a burst of impulses in the larger 'off' fibres which propagate away from the eye. Invasion of the eye via one branch of optic nerve can evoke an answering burst in another branch. 3. Such electrically evoked bursts are similar to light-evoked 'off' bursts with respect to their impulse composition, their ability to be inhibited by illumination of the eye, and their susceptibility to MgCl2 anaesthesia. 4. Invasion of the eye by a train of repetitive electrically evoked impulses in the absence of photic stimulation can give rise to repetitive 'off' bursts as well as concomitant oscillatory potentials in the eye which are similar to those normally evoked by cessation of a photic stimulus. 5. The electrically evoked 'off' bursts appear to be caused by an excitatory rebound following the cessation of inhibitory synaptic input from photoreceptors which can be antidromically activated by electrical stimulation of the optic nerve. 6. The experimental results suggest that the rhythmic discharge of the 'off' fibres evoked by the cessation of a photic stimulus is mediated by the abrupt decrease of inhibitory synaptic input from the receptors.