Rat Optic Nerve Research Articles

Effects of minocycline and tetracycline on retinal ganglion cell survival after axotomy

In the present study, we compared the in vivo neuroprotective efficacy of intraperitoneally administered tetracycline and minocycline to enhance the survival of retinal ganglion cells (RGCs) following unilateral axotomy of the adult rat optic nerve. We also examined the effects of the tetracycline drugs on the activation of retinal microglia. RGCs in retinal whole-mounts were visualized by retrograde labeling with fluorogold. The presence of activated microglia was confirmed immunohistochemically using OX-42 monoclonal antibodies. Optic nerve axotomy produced RGC death and increased activation of microglia. No significant RGC loss was seen prior to 5 days and approximately 50% and 80–90% cell loss occurred at 7 and 14 days, respectively. Examination of the effects of tetracycline and minocycline on RGC survival at 7 days post-axotomy, revealed increased numbers of RGCs in minocycline-treated animals (75% of non-axotomized control) compared with vehicle-only (52% of control) and tetracycline-treated (58% of control) animals. The densities of RGCs (RGCs/mm 2±S.D.) for control, vehicle-, tetracycline- and minocycline-treated axotomized animals were 1996±81, 1029±186, 1158±190 and 1497±312, respectively. The neuroprotective effect of minocycline seen at 7 days was transient, since RGCs present in minocycline-treated animals at 14 days post-axotomy (281±43, 14% of control) were not significantly different to vehicle-treated animals (225±47, 11% of control). OX-42 staining of activated retinal microglia was reduced in tetracycline- and minocycline-treated axotomized animals compared with axotomized animals receiving vehicle-only. These results demonstrate that systemic administration of the second-generation tetracycline derivative, minocycline, delays the death of axotomized RGCs by a mechanism that may be associated with inhibition of microglia activation. The neuroprotective efficacy of minocycline following optic nerve axotomy was superior to that of tetracycline.

CSF from MS patients can induce acute conduction block in the isolated optic nerve

In the present in vitro electrophysiological study, the acute effects of the cerebrospinal fluid (CSF) from multiple sclerosis (MS) and control subjects were measured on the axonal conduction of rat optic nerve, a central tract that is commonly affected in MS. Optic nerve compound action potential (CAP) amplitude was insensitive to the application of CSF obtained from the whole population of non-MS patients and from seven of 15 MS CSF. In the remaining eight MS cases, conversely, a time-dependent depression of CAP amplitude was observed. The reversible blockade of ion channels by soluble substances might account, at least in part, for the transient symptoms often seen in MS patients.

A technique for producing demyelination of the rat optic nerves

Demyelination is a major cause of neurological disability within the human population, and the end product of a number of pathological processes, though the most common is the disease multiple sclerosis. This disease commonly affects the optic pathway, particularly the optic nerve and chiasm. This paper reports a novel technique of producing chemical glial toxin induced demyelination in the rat optic nerves and chiasm by the direct instillation of the glial toxin ethidium bromide into the rat cranial subarachnoid space causing demyelination of the optic nerves and chiasm. This model, because of its involvement of the optic pathways, will allow both histological and functional assessments of the processes of demyelination and remyelination in the rat animal model.

Stimulated regeneration of the crushed adult rat optic nerve correlates with attenuated expression of the protein tyrosine phosphatases RPTPα, STEP, and LAR

We have evaluated the spatial and temporal expression patterns of three protein tyrosine phosphatases (PTPs), receptor PTPα (RPTPα), striatal enriched phosphatase (STEP), and leucocyte common antigen-related phosphatase (LAR), in the retina and optic nerve (ON) of adult rats in which the crushed ON was either regenerating after retinal ganglion cell (RGC) stimulation with intravitreal peripheral nerve (PN) grafting or lens injury (LI), or not regenerating (no treatment). In intact adult rats, all three PTPs were expressed by RGCs and ON glia. In both the regenerating and non-regenerating models, a postlesion rise in RPTPα, STEP, and LAR expression occurred in the RGC somata and in the ON. However, for RPTPα and LAR in the RGCs, and for RPTPα, STEP, and LAR in the ON, this postlesion increase was attenuated in the regenerating versus the non-regenerating models. ON PTP expression changes were localized to glia in the proximal and distal stumps, and to macrophages and extracellular matrix of the glial scar at the lesion site. Interestingly, neither RPTPα, STEP, nor LAR localized to intact or regenerating axons. One explanation of these findings is that RPTPα and LAR may modulate RGC survival, and that RPTPα, STEP, and LAR may modulate axon growth.

Optic nerve regeneration in polyglycolic acid-chitosan conduits coated with recombinant L1-Fc.

Autografts have been extensively studied to facilitate optic nerve (ON) regeneration in animal experiments, but the clinical application of this approach to aid autoregeneration has not yet been attempted. This study aims to explore the guided regeneration by an artificial polyglycolic acid-chitosan conduit coated with recombinant L1-Fc. Consistent with previous studies; in vitro assay showed that both chitosan, a natural biomaterial, and the neural cell adhesion molecule L1-Fc enhanced neurite outgrowth. Rat optic nerve transection was used as an in vivo model. The implanted PGA-chitosan conduit was progressively degraded and absorbed, accompanied by significant axonal regeneration as revealed by immunohistochemistry, anterograde and retrograde tracing. The polyglycolic acid-chitosan conduit coated with L1-Fc showed more effective to promote axonal regeneration and remyelination. Taken together, our observations demonstrated that the L1-Fc coated PGA-chitosan conduits provided a compatible and supportive canal to guild the injured nerve regeneration and remyelination.

Exposure of rat optic nerves to nitric oxide causes protein S-nitrosation and myelin decompaction.

This study investigates the effect of nitric oxide (NO) on both the chemical modifications of CNS proteins and the architecture of the myelinated internode. Incubation of rat optic nerves for 2 h with 1 mM concentration of the NO-donors S-nitroso-N-acetyl-penicillamine (SNAP), ethyl-2-[hydroxyimino]-5-nitro-3-hexeneamide (NOR-3), and 4-phenyl-3-furoxan carbonitrile (PFC) led to decompaction of myelin at the level of the intraperiod line (IPL). In contrast, incubation with 1 mM sodium nitroprusside, which slowly releases NO, sodium nitrite, and N-nitrosopyrrolidine failed to cause myelin disassembly. This suggests that free NO and/or some of its direct oxidation products (e.g., N2O3) are the active molecular species. NO-induced alterations in myelin architecture could not be assigned to protein or lipid degradation, lipid peroxidation, ATP depletion, calcium uptake, protein nitration, protein carbonylation, and nerve depolarization. NO-treatment, however, resulted in the S-nitrosation of a number of proteins. In myelin, one of the major S-nitrosated substrates was identified as proteolipid protein (PLP), an abundant cysteine-rich protein that is responsible for IPL stabilization. Peripheral nervous system myelin, whose stability depends on proteins other than PLP, was not decompacted upon incubation of sciatic nerves with SNAP. It is proposed that NO-mediated nitrosation of sulfhydryl groups is likely to interfere with the normal function of PLP and other important CNS myelin proteins leading to the structural demise of this membrane. These findings are relevant to multiple sclerosis and other inflammatory demyelinating disorders where both excessive NO production and myelin instability are known to occur.

A real-time quantitative PCR comparative study between rat optic and sciatic nerves: determination of neuregulin-1 mRNA levels

Injured axons from peripheral nervous system (PNS) possess the ability to regenerate. In contrast, regeneration of injured axons does not occur in the central nervous system (CNS) or occurs to a limited extent. Previous works have shown that rat sciatic nerve conditioned medium (CM) produced PC12 cells neuronal-like differentiation and neurite outgrowth. In the present work, we compared the expression of neuregulin-1s (NRG-1s) from rat sciatic and optic nerves as members of the PNS and CNS, respectively. Sciatic nerve CM showed a higher neurotrophic activity on PC12 cells than rat optic nerve CM. RT-PCR analysis verified the presence of all three types of NRG-1 mRNAs and their receptors in both types of nerves. Real-time quantitative PCR (QPCR) assays showed that the relative expression levels of all three types of NRG-1 mRNAs were higher in optic nerves than in sciatic nerves. Eleven-day cultured optic nerves showed an increased in NDF and SMDF when compared to freshly isolated optic nerves, whereas GGF decreased. However, 11-day-cultured sciatic nerves only showed an increase in SMDF mRNA. Western blots corroborated the differences in NRG-1 expression profile for both types of nerves and their CMs. Incubation of both CMs with the anti-pan-NRG-1 antibody showed that the neurotrophic activity of the optic nerve CM increased, whereas the sciatic nerve CM remained unchanged. These results indicated that different NRG-1 levels are expressed upon nerve degeneration and the balance between those levels and other neurotrophic factors could have an important role on nerve regeneration.

Glutamate transporter localization does not correspond to the temporary functional recovery and late degeneration after acute ocular ischemia in rats

Purpose To determine whether the localization of retinal glutamate transporters is affected by retinal ischaemia and whether their ability to transport glutamate decreases with the progression of ischemic retinal and optic nerve degeneration. Methods Retinal ischemia was induced in rats by acutely increasing the intraocular pressure (IOP, 110 mmHg/60 min). Reperfusion was permitted for periods up to 60 days post-ischemia. Functional evaluation was performed by monitoring the pupil light reflexes (PLRs) and electroretinograms (flash, flicker ERG and oscillatory potentials). Glutamate transporter localization and d-aspartate (glutamate analogue) uptake were assessed by immunohistochemistry. Results Intense immunoreactivity for the retinal glutamate transporters (GLAST, GLT1, EAAC1 and EAAT5) was observed at all time points after the insult, despite severe retinal degeneration. d-aspartate was also normally accumulated in the ischemic retinas. Ten days post-operatively the PLR ratio (ratio=indirect/direct PLR=34±7·5%) was significantly less than the pre-operative value (pre-op=76·7±2·6%, p<0·05). However, 25 and 35 days post-operatively PLR ratios did not differ significantly from pre-operative values (44·4±6·9 and 53·8±9·6%, p>0·05). Forty-five and 60 days post-operatively the PLR ratio declined again and was significantly lower than the pre-operative value (33·8+8·7 and 26·2+8·9%, p<0·05). Statistical analysis revealed that all tested ERG components had significantly higher values at 32, but not at 42 and 58 days post-operatively when compared to the first time point recorded post-operatively (10 days). Conclusions While retinal glutamate transport is compromised during an acute ischemic insult, consequent retinal recovery and degeneration are not due to a change in the excitatory amino acid transporter localization or d-aspartate (glutamate analogue) uptake. Rat retina and optic nerve are capable of spontaneous, but temporary, functional recovery after an acute ischemic insult.

Acute ischemic injury of astrocytes is mediated by Na-K-Cl cotransport and not Ca2+ influx at a key point in white matter development.

Cerebral palsy is a common birth disorder that frequently involves ischemic-type injury to developing white matter (WM). Dead glial cells are a common feature of this injury and here we describe a novel form of acute ischemic cell death in developing WM astrocytes. Ischemia, modeled by the withdrawal of oxygen and glucose, evoked [Ca2+]i increases and cell death in astrocytes in post-natal day 10 (P10) rat optic nerve (RON). Removing extracellular Ca2+ prevented increases in [Ca2+]i but increased the amount of cell death. Astrocytes showed rapid [Na+]i increases during ischemia and cell death was reduced to control levels by substitution of extracellular Na+ or Cl- or by perfusion with bumetanide, a selective Na-K-Cl cotransport (NKCC) blocker. Astrocytes showed marked swelling during ischemia in the absence of extracellular Ca2+, which was blocked by bumetanide. Raising the extracellular osmolarity to limit water uptake reduced ischemic astrocyte death to control levels. Ultrastructural examination showed that post-ischemic astrocytes had lost their processes and frequently were necrotic, effects partially prevented by bumetanide. At this point in development, therefore, NKCC activation in astrocytes during ischemia produces an osmo-regulatory challenge. Astrocytes can subsequently regulate their cell volume in a Ca2+-dependent fashion but this will require ATP hydrolysis and does not protect the cells against acute cell death.

Mechanism of acute ischemic injury of oligodendroglia in early myelinating white matter: the importance of astrocyte injury and glutamate release.

In developing CNS white matter (WM), the period of early myelination is characterized by a heightened sensitivity to ischemic injury. Using an in situ (isolated) preparation, we show that the mechanism of acute ischemic injury of immature WM oligodendroglial involves Ca2+ influx though non-NMDA type glutamate receptors (GluRs). The Ca2+-influx and acute cell death that was evoked by ischemic conditions (oxygen and glucose withdrawal) in identified P10 rat optic nerve oligodendroglia were blocked by removing extracellular Ca2+ or by CNQX, a selective non-NMDA GluR antagonist. The selective Na-K-Cl cotransport (NKCC) inhibitor bumetanide was also highly protective, even though NKCC expression is restricted to astrocytes in this tissue. Bumetanide largely prevented the non-NMDA GluR-mediated [Ca2+]i rise evoked by ischemia in oligodendroglia, suggesting that it interfered with ischemic glutamate release. In control WM, glutamate-like reactivity was located mainly in astrocytes and oligodendroglia identified using ultrastructural criteria. In ischemic WM, astrocyte glutamate-like reactivity was reduced, an effect countered by bumetanide. We suggest a model in which NKCC-dependent injury and release of glutamate from astrocytes activates glutamate receptors on oligodendroglia, resulting in Ca2+-influx and acute cell death.

Glycogen phosphorylase isozyme pattern in mammalian retinal Müller (glial) cells and in astrocytes of retina and optic nerve.

Müller cells, the radially oriented dominant macroglial cells of the retina, are known to contain abundant glycogen as well as the key enzyme for its degradation, glycogen phosphorylase (GP), but the expressed isozyme pattern is unknown. To elucidate the isoform expression pattern, specific antisera directed against the brain (BB) and muscle (MM) isoforms of GP were applied to retinal sections, isolated Müller cells, and sections of the optic nerve. We show that Müller cells of rat, rabbit, guinea pig, and mouse retina exclusively express the BB isoform. Astrocytes of rat and rabbit optic nerve, as well as retina express only the BB isoform. In contrast, astrocytes in the brain and spinal cord as well as the epithelial cells of the pars caeca and of the ciliary body express both the BB and MM isoform. This result may indicate some differences in the role of glycogen in retinal macroglia and brain astrocytes, reflecting a local specialization of macroglia in the retina proper.

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 histoarchitectural structure of astrocytes which surrounds axons and provides a permissive environment for axon development.

Developmental changes of aggrecan, versican and neurocan in the retina and optic nerve

We have used a monoclonal antibody to neurocan and specific polyclonal antibodies to the non-homologous glycosaminoglycan attachment regions of aggrecan and mRNA splice variants of versican to compare the localization and developmental changes of these structurally related hyaluronan-binding chondroitin sulfate proteoglycans in the rat retina and optic nerve. Staining for aggrecan and versican was first seen at embryonic day 16 in the optic nerve and retina, whereas neurocan was not detected in the embryonic eye. At postnatal day 0 (P0), β-versican staining is largely confined to the inner plexiform layer whereas α-versican is also apparent in the neuroblastic layer. Both aggrecan and, much more weakly, neurocan immunoreactivity is present throughout the neonatal retina. At P9, aggrecan and versican immunoreactivity is most intense in the inner and outer plexiform and ganglion cell layers, accompanied by diffuse staining in the inner and outer nuclear layers. Aggrecan and α-versican are also present throughout the optic nerve and disk, whereas β-versican and neurocan are confined to the laminar beams of the optic nerve. Between P0 and P9 there is a marked increase in β-versican expression in the inner and outer nuclear layers and in the outer plexiform layer, whereas there is only weak staining of neurocan in the inner plexiform and ganglion cell layers of P9 retina. By 1 month postnatal the staining pattern of the fully differentiated retinal layers is essentially identical to that seen in the adult, where there is strong aggrecan and α-versican immunoreactivity in the retina and optic nerve, whereas β-versican has essentially disappeared from the adult retina and, similarly to neurocan, is present only in the laminar beams of the optic nerve. The marked decrease of β-versican in the retina is consistent with >90% decrease in its concentration in brain during postnatal development, suggesting that the developmental time-course for these proteoglycans in retina parallels that seen in other areas of the central nervous system.

Alterations in neurofilament light in optic nerve in rat kainate and monkey ocular hypertension models

The purpose of this study is to evaluate the changes in neurofilament light (NF-L) protein in the optic nerve in rat kainate and monkey ocular hypertension models. In the rat model, optic nerve damage was induced by kainate injection into the vitreous body. In the monkey model, photocoagulation of the trabecular meshwork led to laser-induced ocular hypertension. NF-L in optic nerve extract was quantified by quantitative immunoblot using an imaging analyzer. The amount of NF-L in optic nerve was compared between normal and kainate-treated groups at 7 days after kainate injection. Specimens from rat optic nerves and retinas were evaluated histologically to examine the correlations between damage and amount of NF-L in the optic nerve. In monkeys, the amount of NF-L in the optic nerve was compared between control fellow and ocular hypertensive eyes. Injection of kainate induced morphological optic nerve and retinal damage in rats. The amount of NF-L in the optic nerve was significantly reduced in kainate-treated eyes (vs. normal eyes). The amount of NF-L correlated with the cell count in the ganglion cell layer and the axon number in the optic nerve at 7 days after kainate injection. Further, 6-cyano-7-nitro-quinoxaline-2,3-dione, a non N-methyl- d-aspartate receptor antagonist, suppressed the kainate-induced reduction in NF-L and retinal damage. In the monkey model, ocular hypertension and morphological optic nerve damage were shown by laser-treated eyes. The amount of NF-L in the optic nerve was reduced in laser-treated eyes. In conclusion, in rat kainate and monkey ocular hypertension models, immunoblot evaluation of NF-L shows the reduction of the amount of NF-L in the optic nerves of treated-eyes. The amount of NF-L correlated with the morphological retinal and optic nerve damage in rats. These findings indicate that immunoblot evaluation of NF-L in the optic nerve may provide a quantitative index of optic nerve damage.

Kir4.1 expression by astrocytes and oligodendrocytes in CNS white matter: a developmental study in the rat optic nerve.

Deletion studies in transgenic mice indicate that the potassium inward rectifying channel Kir4.1 is crucial for oligodendrocyte differentiation and has a special role in regulation of extracellular potassium (K(+)), a major function of astrocytes. However, there are conflicting reports on whether Kir4.1 is expressed by white matter astrocytes and oligodendrocytes, raising doubts over its functions. Here, we have examined Kir4.1 expression in astrocytes and oligodendrocytes of the rat optic nerve, a typical central nervous system white matter tract. Single and double immunofluorescence labelling was performed on frozen sections from optic nerves aged postnatal day (P)5, 10, 15, 20 and adult, using anti-Kir4.1 antibodies and the glia-specific antibodies glial fibrillary acidic protein (GFAP, astrocytes), carbonic anhydrase II (CAII, oligodendrocyte somata and processes) and myelin basic protein (MBP, oligodendrocyte myelin sheaths). The results demonstrate Kir4.1 expression in rows of glial cells as early as P5, and this pattern persisted throughout development and into adulthood, consistent with early expression of Kir4.1 on developing oligodendrocytes. Clear co-expression of Kir4.1 and GFAP is first evident at P10 and increases to adult levels by P15 and P20, which correlates with the development of K(+) regulation between P15 and P20. Astrocyte expression of Kir4.1 is localized to perivascular end-feet and fine processes within the fascicles of myelinated axons, consistent with a role in K(+) spatial buffering between nodes of Ranvier and blood vessels. By contrast, Kir4.1 is concentrated in the cell bodies of oligodendrocytes, and there is no apparent co-expression with MBP(+) myelin sheaths, suggesting oligodendroglial Kir4.1 channels are not involved in K(+) regulation. The results support roles for Kir4.1 in both oligodendrocyte differentiation and K(+) regulation by astrocytes.

Canaux ioniques et démyélinisation : les fondements d’un traitement de l’encéphalomyélite autoimmune expérimentale (EAE) par des bloqueurs des canaux potassium

Voltage-gated potassium channels (Kv channels) are ion channels, openings of which provide an outward flow of potassium ions repolarising the cell. In neurons, Kv channels play a crucial role in action potential repolarisation and in shaping neuronal excitability. In non-excitable cells, such as T lymphocytes, Kv channels and calcium-activated K+ channels (KCa channels) determine the driving force for Ca2+ entry. During T cell activation the calcium entry depolarises the cell and increases the cytosolic calcium concentration, which in return activates Kv and KCa channels. K+ channel opening repolarises the cell and drives the membrane potential to a negative voltage. The roles of Kv channels in nervous and immune systems have been investigated here by means of a rat experimental autoimmune disease of the central nervous system, the experimental autoimmune encephalomyelitis (EAE). EAE is characterised clinically by paralysis, and pathologically by inflammatory cell infiltrations into the brain and the spinal cord. Among the inflammatory cells, T lymphocytes play a major role. Hence, EAE can be adoptively transferred into syngenic animals by the injection of T cells reactive to myelin antigens. During adoptive-EAE, somato-sensory evoked potentials recorded along the spinal tracts decrease in amplitude and axonal propagation is disrupted. We have analysed the consequences of Kv channels blockade by peptidyl toxins on central nerve conduction, on T cell activation and on the time course of EAE. In rat optic nerves, Kv channels have been identified up from postnatal day 1. Their blockade by kaliotoxin (a scorpion toxin) or by dendrotoxin-I (a snake toxin) enlarges the compound action potentials, demonstrating the participation of Kv channels to spike repolarisation. This effect disappears at adult age due to the sequestration of Kv channels under the myelin, in the paranodal regions. During acute demyelination by lysophosphatidyl-choline, the surface area of compound action potential decreased probably because conduction block occurred. Demyelination unmasked Kv channels, which are again accessible to toxins. Their blockade by dendrotoxin-I or kaliotoxin favoured a slow delayed conduction suggesting that those Kv channel blockers exert a neurological benefit during demyelinating diseases. In a T-cell line reactive to myelin basic protein antigen, which is used to adoptively transfer experimental autoimmune encephalomyelitis, Kv1.3 channels are constitutively expressed. Their blockade leads to a pronounced reduction of the T cell proliferative response, cytokine production and Ca2+ influx. In the rat, blockade of Kv1.3 inhibits the delayed type hypersensitivity response to myelin basic protein prevents and treats adoptive experimental autoimmune encephalomyelitis. Blockade of Kv channels alone or in combination with KCa channels improves the symptoms of the disease. These results demonstrate that K+ channel blockers displaying high selectivity are potent immunosuppressive agents with beneficial symptomatic effects in experimental autoimmune encephalomyelitis.

Functional evaluation of retina and optic nerve in the rat model of chronic ocular hypertension

Purpose. To functionally characterize the rat retina and optic nerve after chronic elevation of the intraocular pressure (IOP) using electroretinography (ERG) and computerized pupillometry. Methods. Chronic elevation of the IOP was induced in Brown Norway rats by combined injection of indocyanine green dye (ICG) into the anterior chamber and diode laser treatment, followed by ERG and pupil light reflex (PLR) monitoring. Results. Laser treatment induced significant elevation of the IOP in operated eyes for 6 weeks, with maximal values observed 14 days postoperatively (ctrl=18·4±2·4 and operated=35±8·4 mmHg; mean± sd). Preoperative values for the PLR ratio were 68·5±4% (mean± sem; %). Three days postoperatively the PLR ratio decreased to 60·3±10·3%, but was not significantly different compared to preoperative values ( p>0·05, Kruskal-Wallis non-parametric test with Dunn's post-test). However, 7, 14 and 21 days postoperatively the PLR function dramatically decreased to 14·6+8·6, 11·5±6·7 and 12·6±4%, respectively, and was significantly smaller compared to preoperative values ( p<0·01). At day 28 the PLR significantly recovered and was not significantly different compared to preoperative values (PLR ratio=38·5±8·6, p>0·05). However, 35 days after surgery the PLR started to decrease once again in the operated eyes (PLR ratio=17·2±7·4%) and was significantly smaller again compared to preoperative values ( p<0·05). The PLR values continued to decrease until the end of experiment (60 days postoperatively). ERG analysis of operated eyes revealed significantly decreased amplitudes of a- and b-waves 10d postoperatively, while oscillatory potentials (OPs) and flicker ERG (flERG) amplitudes were not detectable. However, 28 days postoperatively OPs significantly, but temporarily recovered, while a-wave, b-wave and flERG amplitudes did not significantly change compared to values observed 10d postoperatively. The ERG analysis of the operated eyes revealed significantly reduced amplitudes 60 days postoperatively. Histological analysis revealed degeneration of all retina layers and optic nerve axons. Conclusions. Chronic ocular hypertension in rats produces dramatic damage to all retinal layers and optic nerves observed by morphological and functional methods which significantly correlate with the IOP elevation. Outer retina of glaucomatous rats seems to be more susceptible to the damage due to chronic elevation of the IOP. Chronic hypertensive rat eyes have capacity to temporarily recover function of the inner retina and optic nerve.

A novel neuroprotectant against retinal ganglion cell damage in a glaucoma model and an optic nerve crush model in the rat.

To investigate the effects of repeated treatments with a neuroprotective compound, R(-)-1-(benzo [b] thiophen-5-yl)-2-[2-(N, N-diethylamino) ethoxy] ethanol hydrochloride (T-588), on retinal ganglion cell (RGC) survival in rat eyes with elevated intraocular pressure (IOP) or after optic nerve crush. An increase in IOP was induced by a single laser treatment to the trabecular meshwork in one eye of adult Wistar rats. Crush injury was unilaterally produced by clipping the optic nerve 2 mm behind the globe. RGC density was estimated by counting fluorescent dye-labeled cells in the flatmount of the retina. The optic nerve damage in the crush model was also evaluated histologically. In the elevated IOP model, RGC survival decreased to 72.9% +/- 3.8% (mean +/- SEM) of that of the contralateral control eye on the eighth day after laser irradiation. Repeated treatments with T-588 at 30 mg/kg twice daily significantly enhanced RGC survival (86.0% +/- 2.2%, P = 0.0242) without the reduction of IOP. In the optic nerve crush model, RGC survival diminished to 37.2% +/- 8.4% of that of the contralateral control eye after 4 weeks. Repeated applications with T-588 at 10 mg/kg twice daily significantly enhanced RGC survival (77.8% +/- 2.1%, P = 0.0038). Histologically, the rat optic nerve in the group treated with T-588 at 10 mg/kg retained a near-normal morphology. T-588 has a neuroprotective effect against RGC death caused by elevated IOP and optic nerve crush in the rat.

Roles for p53 and p73 during oligodendrocyte development.

Oligodendrocytes make myelin in the vertebrate central nervous system (CNS). They develop from oligodendrocyte precursor cells (OPCs), most of which divide a limited number of times before they stop and differentiate. OPCs can be purified from the developing rat optic nerve and stimulated to proliferate in serum-free culture by PDGF. They can be induced to differentiate in vitro by either thyroid hormone (TH) or PDGF withdrawal. It was shown previously that a dominant-negative form of p53 could inhibit OPC differentiation induced by TH but not by PDGF withdrawal, suggesting that the p53 family of proteins might play a part in TH-induced differentiation. As the dominant-negative p53 used inhibited all three known p53 family members - p53, p63 and p73 - it was uncertain which family members are important for this process. Here, we provide evidence that both p53 and p73, but not p63, are involved in TH-induced OPC differentiation and that p73 also plays a crucial part in PDGF-withdrawal-induced differentiation. This is the first evidence for a role of p73 in the differentiation of a normal mammalian cell.

Nicotinic acetylcholine receptors in mouse and rat optic nerves.

Receptor-mediated calcium signaling in axons of mouse and rat optic nerves was examined by selectively staining the axonal population with a calcium indicator. Nicotine (1-50 microM) induced an axonal calcium elevation that was eliminated when calcium was removed from the bath, suggesting that nicotine induces calcium influx into axons. The nicotine response was blocked by d-tubocurarine and mecamylamine but not alpha-bungarotoxin, indicating the presence of calcium permeable, non-alpha7 nicotinic acetylcholine receptor (nAChR) subtype. Agonist efficacy order for eliciting the axonal nAChR calcium response was cytisine approximately nicotine >> acetylcholine. The nicotine-mediated calcium response was attenuated during the process of normal myelination, decreasing by approximately 10-fold from P1 (premyelinated) to P30 (myelinated). Nicotine also caused a rapid reduction in the compound action potential in neonatal optic nerves, consistent with a shunting of the membrane after opening of the nonspecific cationic nicotinic channels. Voltagegated calcium channels contributed little to the axonal calcium elevation during nAChR activation. During repetitive stimulations, the compound action potential in neonatal mouse optic nerves underwent a gradual reduction in amplitude that could be partially prevented by d-tubocurarine, suggesting an activity-dependent release of acetylcholine that activates axonal AChRs. We conclude that mammalian optic nerve axons express nAChRs and suggest that these receptors are activated in an activity-dependent fashion during optic nerve development to modulate axon excitability and biology.