Novel use of a chemically modified siRNA for robust and sustainable in vivo gene silencing in the retina.

Despite efficient and specific in vitro knockdown, more reliable and convenient methods for in vivo knockdown of target genes remain to be developed particularly for retinal research. Using commercially available and chemically modified siRNA so-called Accell siRNA, we established a novel in vivo gene silencing approach in the rat retina. siRNA designed for knockdown of the house keeping gene Gapdh or four retinal cell type-specific genes (Nefl, Pvalb, Rho and Opn1sw) was injected into the vitreous body, and their retinal mRNA levels were quantified using real-time PCR. Intravitreal injection of siRNA for Gapdh resulted in approximately 40-70% reduction in its retinal mRNA levels, which lasted throughout a 9-day study period. Furthermore, all the selected retinal specific genes were efficiently down-regulated by 60-90% following intravitreal injection, suggesting injected siRNA penetrated into major retinal cell types. These findings were consistent with uniform distribution of a fluorescence-labeled siRNA injected into the vitreous body. Interestingly, gene silencing of Grin1, a core subunit of NMDA receptor, was accompanied by significant prevention from NMDA-induced retinal ganglion cell death. Thus, we provide single intravitreal injection of Accell siRNA as a versatile technique for robust and sustainable in vivo retinal gene silencing to characterize their biological functions under physiological and pathophysiological conditions.

Rotenone-induced inner retinal degeneration via presynaptic activation of voltage-dependent sodium and L-type calcium channels in rats.

Rotenone, a mitochondrial complex I inhibitor, causes retinal degeneration via unknown mechanisms. To elucidate the molecular mechanisms of its action, we further characterized a rat model of rotenone-induced retinal degeneration. Intravitreal injection of rotenone (2 nmol/eye) damaged mainly the inner retinal layers, including cell loss in the ganglion cell and inner nuclear layers, which were very similar to those induced by 10 nmol/eye N-methyl-D-aspartate (NMDA). These morphological changes were accompanied by the reduced b-wave amplitude of electroretinogram, and increased immunostaining of 2,4-dinitrophenyl, an oxidative stress marker. Rotenone also downregulated expression of neurofilament light-chain gene (Nfl) as a retinal ganglion cell (RGC) marker. This effect was prevented by simultaneous injection of rotenone with antioxidants or NMDA receptor antagonists. More importantly, voltage-dependent sodium and L-type calcium channel blockers and intracellular calcium signaling modulators remarkably suppressed rotenone-induced Nfl downregulation, whereas none of these agents modified NMDA-induced Nfl downregulation. These results suggest that rotenone-induced inner retinal degeneration stems from indirect postsynaptic NMDA stimulation that is triggered by oxidative stress-mediated presynaptic intracellular calcium signaling via activation of voltage-dependent sodium and L-type calcium channels.

Chemical proteasome inhibition as a novel animal model of inner retinal degeneration in rats.

Chemical proteasome inhibition has been a valuable animal model of neurodegeneration to uncover roles for the ubiquitin-proteasome system in the central nervous system. However, little is known about the effects of chemical proteasome inhibitors on retinal integrity. Therefore, we characterized the effects of structurally different chemical proteasome inhibitors on the retinal morphology and the mechanisms of their action in the normal adult rat eyes. Intravitreal injection of MG-262 and other proteasome inhibitors led to inner retinal degeneration. MG-262-induced inner retinal degeneration was accompanied by reduced proteasome activity, increased poly-ubiquitinated protein levels, and increased positive immunostaining of ubiquitin, 20S proteasome subunit and GADD153/CHOP in the retina. Its retinal degenerative effect was also associated with reduced retinal neurofilament light chain gene expression, reflecting retinal ganglion cell death. MG-262-induced neurofilament light chain downregulation was largely resistant to pharmacological modulation including endoplasmic reticulum stress, apoptosis or MAP kinase inhibitors. Thus, this study provides further evidence of roles for the ubiquitin-proteasome system in the maintenance of the retinal structural integrity. Chemical proteasome inhibition may be used as a novel animal model of inner retinal degeneration, including retinal ganglion cell loss, which warrants further analysis of the molecular mechanisms underlying its retinal degenerative effect.

Nafamostat and sepimostat identified as novel neuroprotective agents via NR2B N-methyl-D-aspartate receptor antagonism using a rat retinal excitotoxicity model.

In addition to its role in the treatment of pancreatitis, the serine protease inhibitor nafamostat exhibits a retinal protective effect. However, the exact mechanisms underlying this effect are unknown. In this study, the neuroprotective effects of nafamostat and its orally active derivative sepimostat against excitotoxicity were further characterised in vitro and in vivo. In primary rat cortical neurons, nafamostat completely suppressed N-methyl-D-aspartate (NMDA)-induced cell death. Intravitreal injection of nafamostat and sepimostat protected the rat retina against NMDA-induced degeneration, whereas the structurally related compounds, gabexate and camostat, did not. The neuroprotective effects of nafamostat and the NR2B antagonist ifenprodil were remarkably suppressed by spermidine, a naturally occurring polyamine that modulates the NR2B subunit. Both nafamostat and sepimostat inhibited [3H]ifenprodil binding to fractionated rat brain membranes. Thus, nafamostat and sepimostat may exert neuroprotective effects against excitotoxic retinal degeneration through NMDA receptor antagonism at the ifenprodil-binding site of the NR2B subunit.

Molecular imaging reveals unique degenerative changes in experimental glaucoma.

Experimentally induced changes in the central visual pathway were studied by using positron emission tomography in monkeys with unilateral hypertension glaucoma. In 2-[18F]fluoro-2-deoxy-glucose studies, monocular visual stimulation of the affected eye yielded significantly reduced neural responses in the occipital visuocortical areas. The response reduction was limited to the visual cortex ipsilateral to the affected eye, indicating the unique vulnerability of ipsilateral visual cortex in experimental unilateral glaucoma. In addition, in [11C]PK11195 positron emission tomography and immunohistochemical studies, selective accumulation of activated microglia, a sign of neural degeneration, was found bilaterally in lateral geniculate nuclei. The present findings establish the usefulness of noninvasive molecular imaging for early diagnosis of glaucoma by providing a sharper surrogate end point for an early phase of glaucoma.

Changes in visual fields and lateral geniculate nucleus in monkey laser-induced high intraocular pressure model.

Monkey eyes are useful for ophthalmologic research into eye diseases because their histological and functional properties are very similar to those of humans. The monkey laser-induced high intraocular pressure (IOP) model is a common model for ophthalmologic research, especially into glaucoma. Although several studies using this model have focused on changes in visual field, retinal ganglion cells (RGC), and lateral geniculate nucleus (LGN), clear relationships among these changes in one and the same monkey have not been established. We therefore examined visual field changes, RGC and LGN numbers, and glial fibrous acidic protein (GFAP) immunohistochemistry in the LGN in each of two monkeys. Visual field sensitivity, RGC number, and neuronal density of LGN were all decreased by high IOP. The relationship between loss of RGC and decrease in visual field sensitivity depended on the eccentricity from the fovea. Moreover, LGN immunohistochemistry revealed greater increases in GFAP expression in the layers receiving a neuronal input from the high IOP eye than in those receiving a neuronal input from the contralateral untreated eye. From these results, we suggest that glaucoma may lead to changes in glial function not only in the retina, but also in the visual pathway, and that such central nervous system changes may be a hallmark of neuropathy in glaucoma, as in other neurodegenerative diseases.

Stachybotrydial selectively enhances fibrin binding and activation of Glu-plasminogen.

Stachybotrydial, a triprenyl phenol metabolite from a fungus, has a plasminogen modulator activity selective to Glu-plasminogen. Stachybotrydial enhanced fibrin binding and activation of Glu-plasminogen (2- to 4-fold enhancement at 60-120 microM) but not of Lys-plasminogen. Approximately 1.2-1.6 moles of [3H]stachybotrydial bound to Glu-plasminogen to exert such effects. The selective modulation of the Glu-plasminogen function by stachybotrydial may be related to alteration of its conformational status.

Intravitreal injection of endothelin-1 caused optic nerve damage following to ocular hypoperfusion in rabbits.

The purpose of this study was to investigate the time course of the ocular hypoperfusion, retinal damage, and optic nerve damage induced by intravitreal injection of endothelin-1 (ET-1) in rabbits. ET-1, at 5 pmol (20 microL, twice a week for 2 or 4 weeks), was injected from the pars plana into the posterior vitreous of the right eye. Optic nerve head (ONH) blood flow and retinal artery diameter, together with the neurofilament light chain (NF-L) content, retinal morphology, and axon density of the optic nerve, were evaluated at 2, 4, and 8 weeks after the first injection of ET-1 (n=7 or 8). Tissue blood velocity in ONH was measured using a laser speckle method, and the diameter of major retinal arteries on the rim of the ONH was calculated from fundus photographs by a masked observer. Histological analysis and immunoblot evaluation of NF-L in the optic nerve were performed to evaluate optic nerve damage. At 2 weeks after the first ET-1 injection, tissue blood velocity was decreased by approximately 20% (versus the contralateral eye), and the diameter of retinal arteries had decreased by approximately 40%. These changes were sustained at the same level until 8 weeks after the first ET-1 injection. At 4 and 8 weeks after the first ET-1 injection, the amount of NF-L in the optic nerve was significantly less in the ET-1 treated eyes than in the contralateral eyes. At 8 weeks after the first ET-1 injection, a loss of myelinated axons and increases in gliosis and connective tissue were noted in the optic nerve of the treated eye, and the optic nerve-axon number had decreased significantly (each, versus the untreated eye). Retinal ganglion cells in the retina were not observed any damage at 2, 4, and 8 weeks after ET-1 injection. In conclusion, intravitreal injection of ET-1 induced chronic hypoperfusion in the ONH and retina, which presumably caused decreases in NF-L content and axon number in the optic nerve noted in the later part of the observation period.

Endothelin-1 impairs retrograde axonal transport and leads to axonal injury in rat optic nerve.

The purpose of this study was to examine the effects of endothelin-1 (ET-1) on retrograde axonal transport in the rat optic nerve. Vehicle or ET-1 (0.2, 1, or 5 pmol/eye) were injected into the vitreous body in Sprague-Dawley rats. Retinal vessels were observed, using a fundus camera, before, and at 10 min, 3 days and 7 days after a single intravitreous injection. Two days after the injection, a neuronal tracer, fluoro gold, was administered via the superior colliculi to retrogradely label active retinal ganglion cells (RGCs). Five days after the tracer administration, retrogradely labeled RGCs were evaluated in the flat-mounted retina, and cross sections from each optic nerve were graded for injury by four independent, masked observers. ET-1 at 5 pmol/eye caused a significant constriction of retinal vessels (versus the vehicle-treated group) at 10 min after the injection. Intravitreous injection of ET-1 caused a dose-related decrease in the number of retrogradely labeled RGCs. Injection of 5 pmol/eye ET-1 led to a statistically significant decrease in the number of retrogradely labeled RGCs (versus the vehicle-treated group). ET-1 at 1 and 5 pmol/eye caused histological optic nerve damage (evaluated using a graded scale). The histological optic nerve damage correlated with the number of retrogradely labeled RGCs. In conclusion, a single intravitreous injection of ET-1 impaired retrograde axonal transport in the rat optic nerve and this impairment correlated with the histological optic nerve damage.

Morphometric evaluation of changes with time in optic disc structure and thickness of retinal nerve fibre layer in chronic ocular hypertensive monkeys.

We examined the time course of changes in optic disc structure by means of a scanning laser ophthalmoscope (Heidelberg Retina Tomograph, HRT) in ocular hypertensive (experimental glaucoma) monkeys, and clarified the relationships between the histological RNFL thickness and HRT parameters. Further, the time course of changes in retinal nerve fiber layer (RNFL) thickness in individual eyes was measured using a scanning laser polarimeter with fixed corneal polarization compensator (GDx FCC). In the present study, two separate experiments were carried out. A chronic intraocular pressure (IOP) elevation was induced by laser trabeculoplasty in the left eye in 11 cynomolgus monkeys. In Experiment 1, the HRT and GDx parameters were measured 12 weeks after the laser treatment in 10 eyes in five monkeys. In Experiment 2, the time course of changes in the HRT and GDx parameters was examined before and 1, 3, 4, 5, 6, 8, 10, 12, 14, and 16 weeks after the laser treatment in 12 eyes in six monkeys. The retardation values (thickness parameters) obtained from the GDx were used to derive thickness and ratio parameters in the superior, inferior, nasal and temporal quadrants. Ratio parameters were expressed as a ratio of superior and inferior quadrant to nasal quadrant. After the last measurements, each eye was enucleated, and retinal cross sections were prepared for histological analysis. In the left (hypertensive) eyes, IOP was persistently elevated throughout the observation periods in both Experiments 1 and 2. In the HRT measurements in Experiment 1, seven out of eight global topographic parameters (exception, disc area) were statistically different between the hypertensive and control eyes 12 weeks after the laser treatment. In Experiment 2, the HRT parameters changed in a time-dependent manner, but each of them almost plateaued at about 4 weeks after the laser treatment. Significant correlations were seen between the histological mean RNFL thickness at 1.5 disc diameters from the optic disc margin and the HRT parameters in 21 eyes from 11 monkeys in Experiments 1 and 2. Especially good correlations with histological mean RNFL thickness were seen for the rim volume and cup volume. In Experiment 1, good correlations were found between GDx ratio parameters and histological RNFL thickness in individual right control eyes (n=5). In individual left experimental glaucoma eyes of Experiment 2 (n=6), GDx ratio parameters declined in a time-dependent manner alongside the IOP elevation. In conclusion, alongside the IOP elevation, time-related changes in optic disc topography and RNFL thickness were demonstrated in monkey eyes using HRT and GDx. HRT (rim and cup) parameters showed good correlations with histological RNFL thickness, and significant interrelations.

Nerve fiber layer measurement using scanning laser polarimetry with fixed corneal compensator in normal cynomolgus monkey eyes.

PURPOSE: The purpose of this study was to examine retinal nerve fiber layer thickness in normal cynomolgus monkeys using a scanning laser polarimeter with a fixed corneal compensator (GDx FCC), and to clarify the reproducibility and symmetries (right-left differences) between both eyes for the GDx parameters. METHODS: GDx parameters were measured in 36 normal eyes of 18 cynomolgus monkeys aged 4.0-5.5 years. The retardation values (thickness parameters) at peripapillary and macular areas obtained from the GDx FCC were measured and calculated thickness, ratio, and modulation parameters in the superior and inferior quadrants. Mean and standard deviation (SD), coefficient of variation (CV), and binocular differences were obtained for each parameter from three independent measurements made during a 1-week period. Correlation between both eyes in macular retardation and baseline values, which indicated the combined minimum retardation values for the nasal and temporal quadrants, and between macular retardation and baseline values were analyzed. RESULTS: The intraocular pressure values (mean +/- SD, n = 18) obtained for the right and left eyes were 20.7 +/- 3.8 and 20.0 +/- 3.2 mm Hg, respectively (no significant differences in both eyes). No significant differences between right and left eyes were detected for any GDx parameters. All parameters showed small right-left differences. The CVs (SD/mean x 100) for all parameters were less than 10%. Highly significant correlations were seen between bilateral eyes for macular retardation (r = 0.936, p < 0.0001) or baseline values (r = 0.946, p < 0.0001). A significant correlation (r = 0.883, p < 0.0001) was also seen between macular retardation and baseline values. CONCLUSIONS: Considering individual differences in corneal birefringence, GDx parameters obtained from a GDx FCC may be useful for the objective evaluation of time-related changes in individual eyes or for binocular comparisons in cynomolgus monkeys.

Comparison between monkey and human visual fields using a personal computer system.

To use monkeys as models for eye diseases that may lead to blindness, we need to develop a method to precisely measure its visual field and to understand similarities and differences in visual field properties between monkeys and humans. The visual field of monkey was not measured precisely although the necessity. We established a new system with personal computers for precise measurement of the monkey visual field. Four monkeys and three humans served as subjects. The luminance-contrast sensitivity of the central 24 degrees field was measured while the subject was fixating a small spot. During the measurement, we continuously recorded the eye position, and discarded the data when fixation was broken. Reliability indices demonstrated high and stable behavioral performance by both monkeys and humans. The luminance-contrast sensitivity was highest around the fovea, and declined as eccentricity increased. The blind spot was clearly detected 15 degrees temporally. The overall sensitivity was higher in humans than in monkeys and the sensitivity dropped more sharply in the periphery in monkeys than in humans. We recommend this system as a convenient and reliable way to measure visual functions in monkeys in basic ophthalmologic research or in assessment of the drug effects on the visual field.