Unmyelinated Optic Nerve Research Articles

Regional Gene Expression in the Retina, Optic Nerve Head, and Optic Nerve of Mice with Optic Nerve Crush and Experimental Glaucoma.

A major risk factor for glaucomatous optic neuropathy is the level of intraocular pressure (IOP), which can lead to retinal ganglion cell axon injury and cell death. The optic nerve has a rostral unmyelinated portion at the optic nerve head followed by a caudal myelinated region. The unmyelinated region is differentially susceptible to IOP-induced damage in rodent models and human glaucoma. While several studies have analyzed gene expression changes in the mouse optic nerve following optic nerve injury, few were designed to consider the regional gene expression differences that exist between these distinct areas. We performed bulk RNA-sequencing on the retina and separately micro-dissected unmyelinated and myelinated optic nerve regions from naïve C57BL/6 mice, mice after optic nerve crush, and mice with microbead-induced experimental glaucoma (total = 36). Gene expression patterns in the naïve unmyelinated optic nerve showed significant enrichment of the Wnt, Hippo, PI3K-Akt, and transforming growth factor β pathways, as well as extracellular matrix-receptor and cell membrane signaling pathways, compared to the myelinated optic nerve and retina. Gene expression changes induced by both injuries were more extensive in the myelinated optic nerve than the unmyelinated region, and greater after nerve crush than glaucoma. Changes present three and fourteen days after injury largely subsided by six weeks. Gene markers of reactive astrocytes did not consistently differ between injury states. Overall, the transcriptomic phenotype of the mouse unmyelinated optic nerve was significantly different from immediately adjacent tissues, likely dominated by expression in astrocytes, whose junctional complexes are inherently important in responding to IOP elevation.

Astrocytes of the eye and optic nerve: heterogeneous populations with unique functions mediate axonal resilience and vulnerability to glaucoma.

The role of glia, particularly astrocytes, in mediating the central nervous system's response to injury and neurodegenerative disease is an increasingly well studied topic. These cells perform myriad support functions under physiological conditions but undergo behavioral changes - collectively referred to as 'reactivity' - in response to the disruption of neuronal homeostasis from insults, including glaucoma. However, much remains unknown about how reactivity alters disease progression - both beneficially and detrimentally - and whether these changes can be therapeutically modulated to improve outcomes. Historically, the heterogeneity of astrocyte behavior has been insufficiently addressed under both physiological and pathological conditions, resulting in a fragmented and often contradictory understanding of their contributions to health and disease. Thanks to increased focus in recent years, we now know this heterogeneity encompasses both intrinsic variation in physiological function and insult-specific changes that vary between pathologies. Although previous studies demonstrate astrocytic alterations in glaucoma, both in human disease and animal models, generally these findings do not conclusively link astrocytes to causative roles in neuroprotection or degeneration, rather than a subsequent response. Efforts to bolster our understanding by drawing on knowledge of brain astrocytes has been constrained by the primacy in the literature of findings from peri-synaptic 'gray matter' astrocytes, whereas much early degeneration in glaucoma occurs in axonal regions populated by fibrous 'white matter' astrocytes. However, by focusing on findings from astrocytes of the anterior visual pathway - those of the retina, unmyelinated optic nerve head, and myelinated optic nerve regions - we aim to highlight aspects of their behavior that may contribute to axonal vulnerability and glaucoma progression, including roles in mitochondrial turnover and energy provisioning. Furthermore, we posit that astrocytes of the retina, optic nerve head and myelinated optic nerve, although sharing developmental origins and linked by a network of gap junctions, may be best understood as distinct populations residing in markedly different niches with accompanying functional specializations. A closer investigation of their behavioral repertoires may elucidate not only their role in glaucoma, but also mechanisms to induce protective behaviors that can impede the progressive axonal damage and retinal ganglion cell death that drive vision loss in this devastating condition.

Effects of experimental glaucoma in Lama1nmf223 mutant mice

To identify changes in response to experimental intraocular pressure (IOP) elevation associated with the laminin α1 nmf223 mutation in mice.Laminin mutant (LM) mice (Lama1nmf223) and C57BL/6J (B6) mice in two age groups each (4–5 months and >1 year) underwent intracameral microbead injections to produce unilaterally elevated IOP. We assessed axonal transport block of immunofluorescently labeled amyloid precursor protein (APP) after 3 days and retinal ganglion cell (RGC) axon loss after 6 weeks. Light, electron and fluorescent microscopy was used to study baseline anatomic differences and effects of 3-day IOP elevation in younger LM mice.In younger mice of both LM and B6 strains, elevated IOP led to increased APP block in the retina, prelaminar optic nerve head (preONH), unmyelinated optic nerve (UON), and myelinated optic nerve (MON). APP blockade not significantly different between younger B6 and LM mouse strains. Older LM mice had greater APP accumulation in both control and glaucoma eyes compared to older B6, however, accumulation was not significantly greater in LM glaucoma eyes compared to LM controls. Axon loss at 6 weeks was 12.2% in younger LM and 18.7% in younger B6 mice (difference between strains, p = 0.22, Mann Whitney test). Untreated LM optic nerve area was lower compared to B6 (nerve area, p < 0.0001, t-test). Aberrant axon bundles, as well as defects, thickening and reduplication of pia mater, were seen in the optic nerves of younger LM mice.Axonal transport blockade significantly differed between old B6 and old LM mice in control and glaucoma eyes, and younger LM mice had abnormal axon paths and lower optic nerve area.

Astrocyte heterogeneity within white matter tracts and a unique subpopulation of optic nerve head astrocytes.

Much of what we know about astrocyte form and function is derived from the study of gray matter protoplasmic astrocytes, whereas white matter fibrous astrocytes remain relatively unexplored. Here, we used the ribotag approach to isolate ribosome-associated mRNA and investigated the transcriptome of uninjured fibrous astrocytes from three regions: unmyelinated optic nerve head, myelinated optic nerve proper, and corpus callosum. Astrocytes from each region were transcriptionally distinct and we identified region-specific astrocyte genes and pathways. Energy metabolism, particularly oxidative phosphorylation and mitochondrial protein translation emerged as key differentiators of astrocyte populations. Optic nerve astrocytes expressed higher levels of neuroinflammatory pathways than corpus callosum astrocytes and we further identified CARTPT as a new marker of optic nerve head astrocytes. These previously uncharacterized transcriptional profiles of white matter astrocyte types reveal their functional diversity and a greater heterogeneity than previously appreciated.

The role of aquaporin-4 in optic nerve head astrocytes in experimental glaucoma.

To study aquaporin channel expression in astrocytes of the mouse optic nerve (ON) and the response to IOP elevation in mice lacking aquaporin 4 (AQP4 null). C57BL/6 (B6) and AQP4 null mice were exposed to bead-induced IOP elevation for 3 days (3D-IOP), 1 and 6 weeks. Mouse ocular tissue sections were immunolabeled against aquaporins 1(AQP1), 4(AQP4), and 9(AQP9). Ocular tissue was imaged to identify normal AQP distribution, ON changes, and axon loss after IOP elevation. Ultrastructure examination, cell proliferation, gene expression, and transport block were also analyzed. B6 mice had abundant AQP4 expression in Müller cells, astrocytes of retina and myelinated ON (MON), but minimal AQP4in prelaminar and unmyelinated ON (UON). MON of AQP4 nulls had smaller ON area, smaller axon diameter, higher axon density, and larger proportionate axon area than B6 (all p≤0.05). Bead-injection led to comparable 3D-IOP elevation (p = 0.42) and axonal transport blockade in both strains. In B6, AQP4 distribution was unchanged after 3D-IOP. At baseline, AQP1 and AQP9 were present in retina, but not in UON and this was unaffected after IOP elevation in both strains. In 3D-IOP mice, ON astrocytes and microglia proliferated, more in B6 than AQP4 null. After 6 week IOP elevation, axon loss occurred equally in the two mouse types (24.6%, AQP4 null vs. 23.3%, B6). Lack of AQP4 was neither protective nor detrimental to the effects of IOP elevation. The minimal presence of AQP4 in UON may be a vital aspect of the regionally specific phenotype of astrocytes in the mouse optic nerve head.

The Bioelectric Field of the Pattern Electroretinogram in the Mouse

To compare the bioelectric field associated with the pattern electroretinogram (PERG) with that of the flash electroretinogram (FERG) in the mouse. PERGs and FERGs were recorded from each eye in 32 C57BL/6J mice using corneal silver loops referenced to a subcutaneous needle on the back of the head. PERG stimuli were horizontal gratings of 0.05 cycles per degree and 98% contrast reversing 2 times per second. Light-adapted FERG stimuli were bright strobe flashes. Stimuli were presented either monocularly or binocularly. In some experiments, TTX was injected in one eye and saline in the contralateral eye. The PERG recorded from the contralateral, occluded eye had slightly larger amplitude (1.14 ×, P < 0.01) and longer latency (+1.57 ms, P < 0.01) compared with the ipsilateral eye. Under binocular stimulation, the PERG amplitude was much larger (1.67 ×, P < 0.01) than the monocular amplitude. TTX injected in the stimulated eye drastically reduced the PERG in both eyes. Monocular FERGs were recordable from the stimulated eye only and were moderately reduced by TTX. Binocular and monocular FERGs had similar amplitudes. PERG and FERG generate different bioelectric fields in the mouse. The PERG bioelectric field is consistent with a dipole model whose axis is orthogonal to the eye axis, whereas the standard dipole model for the FERG is coaxial. Possible sources of the PERG bioelectric field are unmyelinated optic nerve axons adjacent to the sclera. Results provide new insights on the generators of the PERG signal and its alterations in mouse models of glaucoma and optic nerve diseases.

Early microglia activation in a mouse model of chronic glaucoma

Changes in microglial cell activation and distribution are associated with neuronal decline in the central nervous system (CNS), particularly under pathological conditions. Activated microglia converge on the initial site of axonal degeneration in human glaucoma, yet their part in its pathophysiology remains unresolved. To begin with, it is unknown whether microglia activation precedes or is a late consequence of retinal ganglion cell (RGC) neurodegeneration. Here we address this critical element in DBA/2J (D2) mice, an established model of chronic inherited glaucoma, using as a control the congenic substrain DBA/2J Gpnmb(+/SjJ) (D2G), which is not affected by glaucoma. We analyzed the spatial distribution and timecourse of microglial changes in the retina, as well as within the proximal optic nerve prior to and throughout ages when neurodegeneration has been reported. Exclusively in D2 mice, we detected early microglia clustering in the inner central retina and unmyelinated optic nerve regions, with microglia activation peaking by 3 months of age. Between 5 and 8 months of age, activated microglia persisted and concentrated in the optic disc, but also localized to the retinal periphery. Collectively, our findings suggest microglia activation is an early alteration in the retina and optic nerve in D2 glaucoma, potentially contributing to disease onset or progression. Ultimately, detection of microglial activation may have value in early disease diagnosis, while modulation of microglial responses may alter disease progression.

Early microglia activation in a mouse model of chronic glaucoma

AbstractChanges in microglial cell activation and distribution are associated with neuronal decline in the central nervous system (CNS), particularly under pathological conditions. Activated microglia converge on the initial site of axonal degeneration in human glaucoma, yet their part in its pathophysiology remains unresolved. To begin with, it is unknown whether microglia activation precedes or is a late consequence of retinal ganglion cell (RGC) neurodegeneration. Here we address this critical element in DBA/2J (D2) mice, an established model of chronic inherited glaucoma, using as a control the congenic substrain DBA/2J Gpnmb+/SjJ (D2G), which is not affected by glaucoma. We analyzed the spatial distribution and timecourse of microglial changes in the retina, as well as within the proximal optic nerve prior to and throughout ages when neurodegeneration has been reported. Exclusively in D2 mice, we detected early microglia clustering in the inner central retina and unmyelinated optic nerve regions, with microglia activation peaking by 3 months of age. Between 5 and 8 months of age, activated microglia persisted and concentrated in the optic disc, but also localized to the retinal periphery. Collectively, our findings suggest microglia activation is an early alteration in the retina and optic nerve in D2 glaucoma, potentially contributing to disease onset or progression. Ultimately, detection of microglial activation may have value in early disease diagnosis, while modulation of microglial responses may alter disease progression. J. Comp. Neurol. 519:599–620, 2011. © 2010 Wiley‐Liss, Inc.

Optic nerve dynein motor protein distribution changes with intraocular pressure elevation in a rat model of glaucoma

Acute intraocular pressure (IOP) elevation causes accumulation of retrogradely-transported brain derived neurotrophic factor and its receptor at the optic nerve head (ONH) in rats and monkeys. Obstruction of axonal transport may therefore be involved in glaucoma pathogenesis, but it is unknown if obstruction is specific to certain transported factors or represents a generalized failure of retrograde axonal transport. The dynein motor complex mediates retrograde axonal transport in retinal ganglion cells (RGC). Our hypothesis was that elevated IOP interferes with dynein-mediated axonal transport. We studied the distribution of dynein subunits in the retina and optic nerve after acute and chronic experimental IOP elevation in the rat. IOP was elevated unilaterally in 54 rats. Dynein subunit distribution was compared in treated and control eyes by immunohistochemistry and Western blotting at 1 day ( n = 12), 3 days ( n = 4), 1 week ( n = 15), 2 weeks ( n = 12) and 4 weeks ( n = 11). For immunohistochemistry, sections through the ONH were probed with an anti-dynein heavy chain (HC) antibody and graded semi-quantitatively by masked observers. Other freshly enucleated eyes were microdissected for separate Western blot quantification of dynein intermediate complex (IC) in myelinated and unmyelinated optic nerve, ONH and retina. Immunohistochemistry showed accumulation of dynein HC at the ONH in IOP elevation eyes compared to controls ( P < 0.001, Wilcoxon paired sign-rank test, n = 29). ONH dynein IC was elevated by 46.5% in chronic IOP elevation eyes compared to controls by Western blotting ( P < 0.001, 95% CI = 25.9% to 67.8%, n = 17). The maximum increase in ONH dynein IC was 78.7% after 1 week ( P < 0.05, n = 5), but significant increases were also detected after 4 h and 4 weeks of IOP elevation ( P < 0.05, n = 4 rats per group). Total retinal dynein IC was increased by 8.7% in chronic IOP elevation eyes compared to controls ( P < 0.03, 95% CI 1.4% to 16.1%, n = 24). In the retina, IOP elevation particularly affected the 72 kD subunit of dynein IC, which was 100.7% higher in chronic IOP elevation eyes compared to controls ( P < 0.00001, 95% CI 71.0% to 130.4%, n = 21). Dynein IC changes in myelinated and unmyelinated optic nerve were not significant ( P > 0.05). We conclude that dynein accumulates at the ONH with experimental IOP elevation in the rat, supporting the hypothesis that disrupted axonal transport in RGC may be involved in the pathogenesis of glaucoma. The effect of IOP elevation on other motor proteins deserves further investigation in the future.

Fine Structure of the Retino-Optic Nerve Junction in Dogs

In most mammals, the optic nerve fibers are myelinated in its extraocular part (EON) but not in its intraocular part (ION) and also in the retina. Transitional zone from the myelinated to unmyelinated optic nerve usually lies in the central part to the lamina cribrosa. It has been known that dogs contain exceptionally myelinated fibers in ION by light microscopy. The aim of this study was to investigate electron microscopically the retino-optic nerve junction in dogs and re-evaluate the barrier to migration of oligodendroblasts into ION. Fourteen adult dogs were used. EON was largely myelinated. In ION the percentage of myelinated fibers decreased gradually toward the retina. A narrow area of ION adjoining the retina was completely unmyelinated. In most mammalian optic nerves, oligodendrocytes are not found in ION. It has been suggested that oligodendroblasts are prevented from migrating from EON into ION; that is to say, there is a barrier to migration of oligodendroblasts. The lamina cribrosa, a dense meshwork of fibrous astrocytic processes, and a defect in the blood optic nerve barrier have been proposed as a candidate for the barrier to migration. Our results suggest, however, that these factors, at least in dogs, would be not involved in the formation of a barrier to migration of oligodendroblasts.

Acid Phosphatase Localization in Accumulated Membranous Organelles of Optic Nerve Axons Following Acute Elevation of Intraocular Pressure

Acid phosphatase localization in accumulated membranous organelles of optic nerve axons of guinea pigs following acute elevation of intraocular pressure (IOP) was determined, employing light and electron microscopic enzymic cytochemistry with β-glycerophosphate as a substrate. Positive reaction products appeared to accumulate in the region of the lamina cribrosa, as revealed with light microscopic enzyme cytochemistry. Electron microscopic enzyme cytochemistry also demonstrated that such reaction products mainly localized on multivesicular or multilamellar bodies and myelin-like structures in the unmyelinated optic nerve axons. Following an acutely elevated IOP, retrograde-moving membranous organelles in the optic nerve axons were found to contain AcPase, suggesting that these organelles could be degraded in the axons through the lysosomal pathway.

Presynaptic calcium dynamics at the frog retinotectal synapse.

1. We characterized the kinetics of presynaptic Ca2+ ion concentration in optic nerve fibers and terminals of the optic tectum in Rana pipiens with the use of microfluorimetry. Isolated frog brains were incubated with the membrane-permeant tetraacetoxymethyl ester (AM) of the Ca2+ indicator fura-2. An optic nerve shock caused a transient decrease in the 380-nm excited fluorescence in the optic tectum with a rise time of <15 ms and a recovery to prestimulus levels on a time scale of seconds. 2. In normal saline, the amplitude of the fluorescence transients was dependent on stimulus intensity and at all levels it was directly correlated with the amplitude of postsynaptic field potentials produced by activation of unmyelinated optic nerve fibers. In the presence of the non-N-methyl-D-aspartate glutamate receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione, the amplitude and time course of fluorescence transients remained essentially unchanged while postsynaptic field potential amplitude was greatly reduced. Replacing extracellular Ca2+ with Ba2+ blocked unfacilitated postsynaptic field potentials while fluorescence transients remained significant. In reduced-Ca2+ salines (<1 mM), the amplitude of fluorescence transients increased approximately linearly with extracellular [Ca2+], whereas the amplitude the corresponding field potential was nonlinearly related to the fluorescent transient amplitude (approximately 2.5 power). In thin sections of labeled tecta, fluorescence labeling was localized to 1-micron puncta in the termination zone of optic nerve fibers in the superficial layers. Taken together, these results provide strong evidence that the fluorescence transients correspond to an increase in Ca2+ in presynaptic terminals of unmyelinated optic nerve fibers. 3. During trains of optic nerve stimulation, the amplitude of fluorescence transients to succeeding action potentials became smaller. The decrement of the amplitudes was not observed in mag-fura-5-labeled tecta, when the intracellular Ca2+ buffering capacity of fura-2-labeled terminals was increased by incubation with bis-(o-aminophenoxy)-N,N,N',N'-tetraacetic acid (BAPTA)-AM or ethylene glycol-bis (beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA)-AM, or in low-Ca2+ saline. We conclude that the Ca2+ influx per action potential is constant during the train and that the reduced response was produced by saturation of the fura-2. We provide a mathematical analysis of this saturation effect and use it to estimate the Ca2+ change per action potential. 4. Both BAPTA-AM and EGTA-AM reduced the overall amplitude of fura-2-measured Ca2+ transients and reduced the saturation effect in action potential trains. However, there was a qualitative difference in their effects on the shape of the transient. Incubation with the fast buffer BAPTA prolonged the decay to baseline. In contrast, the slow buffer EGTA (or EDTA) produced an initial decay faster than the control condition while also producing the slower subsequent phase observed with BAPTA. We demonstrate that these results are consistent with numerical simulations of Ca2+ dynamics in a single-compartment model where the fast initial decay is produced by the forward rate of Ca2+ binding to EGTA. 5. Ca2+ influx into tectal presynaptic structures, and also into unmyelinated axons in the isolated optic nerve, was diminished (60-70%) in the presence of the voltage-activated Ca2+ channel blocker omega-conotoxin GVIA, but was only weakly affected (approximately 10%) by omega-agatoxin IVA. 6. After 10- to 50-Hz stimulus trains, synaptic enhancement of unmyelinated fibers decayed with a characteristic time similar to fura-2 fluorescence decays. Incubation with EDTA-AM or EGTA-AM produced little effect on evoked release but reduced both the amplitude of the fura-2-measured Ca2+ transient and the amplitude of short-term synaptic enhancement.

Localization of NADPH diaphorase/nitric oxide synthase in the optic nerve of the normal guinea pig: A light and electron microscopic study

The aim of this study is to determine the presence and subcellular distribution of NADPH diaphorase (NADPH-d)/nitric oxide synthase (NOS) in the optic nerve of the normal guinea pig. Optic nerve specimens were stained by NADPH-d histochemistry, and double labeled by combining NADPH-d histochemistry with immunostaining for (a) anti-glial fibrillary acidic protein (GFAP) antibody for recognition of astrocytes, (b) griffonia simplicifilia B4-isolectin (GSA-IB4) horse radish peroxidase (HRP)-conjugate for identification of microglia, or (c) oligodendrocyte-associated antibodies to carbonic anhydrase isoenzyme II (CA-II) or to galactocerebroside (GalC) for visualization of oligodendrocytes. In addition, constitutive NOS (cNOS) and inducible NOS (iNOS) immunostaining were used for colocalization with NADPH-d histochemistry. Light microscopy revealed NADPH-d reaction product in the blood vessels and neuroglia of the unmyelinated optic nerve head and myelinated retrobulbar optic nerve. Double labeling with GFAP immunoperoxidase combined with NADPH-d histochemistry revealed both activities in astrocytes. Microglia were labeled with GSA-IB4 isolectin HRP-conjugate, but they did not have NADPH-d activity. Oligodendroglia were immunolabeled with anti CA-II or antiGalC antibodies, but they did not have NADPH-d activity. Both iNOS and cNOS immunoperoxidase labeled astrocytes, but not microglia or oligodendroglia. Under transmission electron microscopy, NADPH-d reaction product appeared as electron-dense particles. These particles were seen in the cytoplasm of endothelial cells, perivascular smooth muscle cells and fibrous astrocytes. Axons and myelin were devoid of NADPH-d activity. This study demonstrates the existence and cellular distribution of NADPH-d/NOS activity in endothelial cells, perivascular smooth muscle cells and fibrous astrocytes of the optic nerve of the normal guinea pig. The presence of these non-neuronal sources of NOS in the optic nerve provides the foundation for future comparative studies of the functional role of reactive oxygen induced toxicity in disorders affecting the optic nerve. © 1996 Wiley-Liss, Inc.

Localization of NADPH diaphorase/nitric oxide synthase in the optic nerve of the normal guinea pig: A light and electron microscopic study

The aim of this study is to determine the presence and subcellular distribution of NADPH diaphorase (NADPH-d)/nitric oxide synthase (NOS) in the optic nerve of the normal guinea pig. Optic nerve specimens were stained by NADPH-d histochemistry, and double labeled by combining NADPH-d histochemistry with immunostaining for (a) anti-glial fibrillary acidic protein (GFAP) antibody for recognition of astrocytes, (b) griffonia simplicifilia B4-isolectin (GSA-IB4) horse radish peroxidase (HRP)-conjugate for identification of microglia, or (c) oligodendrocyte-associated antibodies to carbonic anhydrase isoenzyme II (CA-II) or to galactocerebroside (GalC) for visualization of oligodendrocytes. In addition, constitutive NOS (cNOS) and inducible NOS (iNOS) immunostaining were used for colocalization with NADPH-d histochemistry. Light microscopy revealed NADPH-d reaction product in the blood vessels and neuroglia of the unmyelinated optic nerve head and myelinated retrobulbar optic nerve. Double labeling with GFAP immunoperoxidase combined with NADPH-d histochemistry revealed both activities in astrocytes. Microglia were labeled with GSA-IB4 isolectin HRP-conjugate, but they did not have NADPH-d activity. Oligodendroglia were immunolabeled with anti CA-II or anti GalC antibodies, but they did not have NADPH-d activity. Both iNOS and cNOS immunoperoxidase labeled astrocytes, but not microglia or oligodendroglia. Under transmission electron microscopy, NADPH-d reaction product appeared as electron-dense particles. These particles were seen in the cytoplasm of endothelial cells, perivascular smooth muscle cells and fibrous astrocytes. Axons and myelin were devoid of NADPH-d activity. This study demonstrates the existence and cellular distribution of NADPH-d/NOS activity in endothelial cells, perivascular smooth muscle cells and fibrous astrocytes of the optic nerve of the normal guinea pig. The presence of these non-neuronal sources of NOS in the optic nerve provides the foundation for future comparative studies of the functional role of reactive oxygen induced toxicity in disorders affecting the optic nerve.

Embryonic optic nerve tissue fails to support neurite outgrowth by central and peripheral neurons in vitro.

The failure of axon regeneration in the injured mammalian central nervous system has been ascribed, in part, to the inhibitory effects of myelin proteins. To investigate the influence of myelination on neurite growth and regeneration by both central nervous system and peripheral nervous system neurons, isolated rat neonatal retinal ganglion cells and adult and neonatal dorsal root ganglion neurons were cultured on cryostat sections of both immature unmyelinated and mature fully myelinated adult rat optic nerve. In agreement with earlier studies using neonatal peripheral neurons, the adult optic nerve failed to support neurite outgrowth from any of the neurons tested. A new finding was that tissue sections from unmyelinated optic nerve (aged embryonic days 18 and 20, and postnatal days 1-3), also failed to support the growth of neurites from neonatal retinal ganglion cells and both neonatal and adult dorsal root ganglion neurons. Neonatal retinal ganglion cells also failed to extend neurites on sections of pre-degenerated sciatic nerve, a tissue shown in our previous work to be a good substratum for supporting neurite growth for both neonatal and adult DRG neurons. These results suggest that cells in the immature optic nerve either express widely acting axon growth inhibitory molecules unrelated to previously described myelin proteins, or do not synthesize appropriate axon growth promoting molecules. They also reveal that, for axon regeneration, central nervous system and peripheral sensory neurons require distinct substratum interactions.

Role of hydrogen peroxide in experimental optic neuritis. A serial quantitative ultrastructural study.

In order to determine the role of H2O2 in demyelination of the optic nerve, serial quantitative analysis of H2O2-derived cerium perhydroxide reaction product particles was obtained by computerized digitization of electron micrographs of the myelinated retrobulbar optic nerve, unmyelinated optic nerve head, and the optic nerve sheath of guinea pigs sensitized for experimental allergic encephalomyelitis (EAE) and euthanized 3-14 days later. We found that cerium perhydroxide reaction product particles were greatest in the myelinated optic nerve 3 days after antigenic sensitization, but at this focus decreased 7-14 days after antigenic sensitization. Reaction product accumulated in the unmyelinated optic nerve head and optic nerve sheath 3-14 days after sensitization. These results in the myelinated optic nerve suggest H2O2 consumption results in peroxidation of myelin lipid as demyelination proceeds 7-14 days after antigenic sensitization. Hydrogen peroxide accumulation in the optic nerve head and the optic nerve sheath appears to provide a reservoir for diffusion of H2O2 into the retrobulbar optic nerve and adjacent perineural nerve, contributing to the frequent predilection for optic nerve involvement in EAE and perhaps in multiple sclerosis.

Ultrastructure of Glial Cells and Extracellular Matrix in the Guinea Pig Lamina Cribrosa

The ultrastructure of glial cells and extracellular matrix that support normal guinea pig optic nerve axons at the lamina cribrosa was examined by conventional thin-section transmission electron microscopy and by the quick-freeze, deep-etch, rotary-shadow technique. Glial cell bodies and processes were identified by their characteristic parallel intermediate filaments that were closely packed with reduced cross-linking between adjacent filaments when compared with neighboring axons. Glial processes surrounded lamina cribrosa beams and separated axons from the beams. These beams contained bundles of collagen fibrils. Short regular cross-bridges spanned the spaces between the collagen fibrils much like rungs of a ladder. Surrounding the collagen bundles was a network-like peribeam extracellular matrix. This matrix extended to the basement membrane of the surrounding, stratified processes of glial cells. The glial basement membrane was connected to the glial plasma membrane by numerous orthogonal fibrous strands. In regions within basement membrane there also were cross-bridges from the glial plasma membrane to intermediate filaments within the cell. Apposed glial process membranes appeared to directly contact each other. Unmyelinated optic nerve axon membranes and glial process membranes also directly abutted each other. Overall, the cross-bridging structural organization was dense near collagen beams, and became progressively less dense toward the axon. This organization of the lamina cribrosa may play an important role in the distribution of tissue shear stress associated with intraocular pressure and thus protect the retinal ganglion cell axons as they pass through the lamina cribrosa.

Sources of electrical transients in tectal neuropil of the frog, Rana pipiens

We have studied the outer neuropil layers in frog tectum where the ummyelinated optic nerve fibers terminate. At any point the neuropil an extracellular microelectrode records several different visually evoked electrical transients, distinct by size and shape. When classified by shape alone, each transient falls into one of 3 distinct classes. Some of these transients are binocularly driven, as originally described by Finch and Collett. The aggregate of the receptive fields of all the elements recorded at a single point defines a multiunit receptive field (MURF). Each MURF is characteristically oval, and divided into 3 sections along its long axis. Each section represents the aggregate of the receptive fields associated with one class of transient; i.e. transients belonging to only one specific class can be evoked by stimulating that part of the visual field corresponding to the appropriate section of the MURF. All of the MURFs mapped by recording in a single tectum are radially arranged in visual space about a central point, or ‘visual pole’. Several conclusions are made. First, the two larger types of transient are generated postsynaptically by electrically active dendritic elements, specifically the beaded dendritic appendages of tectal neurons. The smallest type of transient is of presynaptic origin. Second, these tectal elements have a local and global anatomical order across the tectum, which accounts for both the tripartite structure of the MURFs and their radial arrangement about a visual pole. Third, since the large transients are of postsynaptic origin, genuine recordings of single retinal ganglion cell (RGC) activity can be made only in the optic nerve or retina itself. Fourth, information is conveyed over the unmyelinated optic nerve fibers at pulse rates as high as 80/s and is transsynaptically effective at such rates. Finally, the electrically active tectal dendritic elements, with their highly organized spatial arrangement, are an important component of the frog's visual processing apparatus, instead of being merely relays or repeaters.

Immunohistological localization of the adhesion molecules L1, N‐CAM, and MAG in the developing and adult optic nerve of mice

The localization of the cell adhesion molecules L1, neural cell adhesion molecule (N-CAM), and myelin-associated glycoprotein (MAG) was studied immunohistologically at the light and electron microscopic levels and immunochemically in the developing and adult mouse optic nerve and retina. The neural adhesion molecule L1 is strongly expressed on the shafts of fasciculating unmyelinated axons at all ages studied from embryonic day 15 through adulthood. Growth cones of retinal ganglion cell axons were weakly L1-positive or L1-negative when contacting glial cells. Unmyelinated axons were not only L1-positive when contacting each other but also when contacting glia, whereas contacts between glial cells were L1-negative at all developmental unmyelinated retinal nerve fiber layer or in the unmyelinated optic nerve head became L1-negative when enwrapped by myelin in the optic nerve proper. At all stages of development N-CAM showed profuse labeling on fasciculating axons, growth cones, and their contact sites with glial cells as well as contacts between glial cells. In contrast to L1, axons remained N-CAM-positive when becoming myelinated. Sometimes, N-CAM was found in compact myelin. However, N-CAM was absent from glial surfaces contacting basement membranes at the interface to meninges, blood vessels, and the vitreous body of the eye. MAG was first detectable intracellularly in oligodendrocytes associated with the endoplasmic reticulum and Golgi apparatus before it became apparent at the cell surface. There it was present on oligodendrocytes prior and during the first stages of ensheathment of axons, both on cell body and processes. After formation of compact myelin MAG remained strongly expressed periaxonally and was only weakly detectable in noncompacted myelin including inner mesaxon and paranodal loops. None of the adhesion molecules was detectable on extracellular matrix, in the meninges, or on endothelial cells. Immunochemical analysis of antigen expression at different developmental stages was in agreement with the immunohistological data. We infer from these observations that L1 is involved in stabilization not only of axon-axon, but also axon-glia contacts, while the more dynamic structure of the growth cone generally expresses less L1. A differential expression of L1 along the course of an axon--being present on its unmyelinated, but absent on its myelinated part--further supports the notion that L1 may be involved in the stabilization of axonal fascicles but not of axon-myelin contacts.(ABSTRACT TRUNCATED AT 400 WORDS)

Postnatal differentiation of rat optic nerve fibers: electron microscopic observations on the development of nodes of Ranvier and axoglial relations.

The postnatal differentiation of rat optic nerve fibres was examined by transmission electron microscopy. The results show that many early developing axons contain clusters of vesiculotubular profiles prior to myelination. At places vesicular elements appear to fuse with the axolemma, and, in addition, some axons exhibit deep axolemmal invaginations and axoplasmic lamellated bodies. It is suggested that these features might reflect axolemmal remodelling, possibly involving axoglial signalling and/or functional differentiation of the axolemma. The size distribution of unmyelinated optic nerve axons changes little during development. Ensheathment of larger axons commences 6 days postnatally. The subsequent formation of compact myelin sheaths is accompanied by an increase in axonal diameter. The early sheaths are a few microns long and separated by long bare axon segments. In optic nerves from 10-12-day-old rat pups, a few sheaths consisting of about five layers border primitive asymmetric nodes with a patchy axolemmal undercoating. Extensions from one of the terminating sheaths are often associated with undercoated patches of axolemma. Relatively differentiated nodes of Ranvier first appear 14-16 days after birth. The continued nodal maturation involves establishment of a regular nodal geometry, increasing distinctness of the axolemmal undercoating, and formation of perinodal astrocytic processes embedded in an extracellular node gap substance. The results are compared with available data on the conduction properties of rat optic nerve fibres during development.