Duco I. Hamasaki

Rpe65 is necessary for production of 11- cis-vitamin A in the retinal visual cycle

Mutation of RPE65 can cause severe blindness from birth or early childhood, and RPE65 protein is associated with retinal pigment epithelium (RPE) vitamin A metabolism. Here, we show that Rpe65-deficient mice exhibit changes in retinal physiology and biochemistry. Outer segment discs of rod photoreceptors in Rpe65–/– mice are disorganized compared with those of Rpe65+/+ and Rpe65+/– mice. Rod function, as measured by electroretinography, is abolished in Rpe65–/– mice, although cone function remains. Rpe65–/– mice lack rhodopsin, but not opsin apoprotein. Furthermore, all-trans-retinyl esters over-accumulate in the RPE of Rpe65–/– mice, whereas 11-cis-retinyl esters are absent. Disruption of the RPE-based metabolism of all-trans-retinyl esters to 11-cis-retinal thus appears to underlie the Rpe65-/- phenotype, although cone pigment regeneration may be dependent on a separate pathway.

Sulfur Hexafluoride in Owl Monkey Vitreous Cavity

Sulfur hexafluoride (SF6) gas, tested in the vitreous chamber of owl monkey eyes, remained in the vitreous cavity about twice as long as air (ten to 11 days vs. five to six days). Its volume expanded from the diffusion of other gases into the created gas pocket. Lens opacities developed when the lens was in continuous contact with SF6 or air. Sulfur hexafluoride appeared to be no more toxic to the retina than air, using electroretinographic, histologic, and electron microscopic criteria.

Astrocytes react to oligemia in the forebrain induced by chronic bilateral common carotid artery occlusion in rats.

The effects of oligemia (moderate ischemia) on the brain need to be explored because of the potential role of subtle microvascular changes in vascular cognitive impairment and dementia. Chronic bilateral common carotid artery occlusion (BCCAO) in adult rats has been used to study effects of oligemia (hypoperfusion) using neuropathological and neurochemical analysis as well as behavioral tests. In this study, BCCAO was induced for 1 week, or 2, 4, and 6 months. Sensitive immunohistochemistry with marker proteins was used to study reactions of astrocytes (GFAP, nestin), and lectin binding to study microglial cells during BCCAO. Overt neuronal loss was visualized with NeuN antibodies. Astrocytes reacted to changes in the optic tract at all time points, and strong glial reactions also occurred in the target areas of retinal fibers, indicating damage to the retina and optic nerve. Astrocytes indicated a change in the corpus callosum from early to late time points. Diffuse increases in GFAP labeling occurred in parts of the neocortex after 1 week of BCCAO, in the absence of focal changes of neuronal marker proteins. No significant differences emerged in the cortex at longer time points. Nestin labeling was elevated in the optic tract. Reactions of microglia cells were seen in the cortex after 1 week. Measurements of the basilar artery indicated a considerable hypertrophy, indicative of macrovascular compensation in the chronic occlusion model. These results indicate that chronic BCCAO and, by inference, oligemia have a transient effect on the neocortex and a long-lasting effect on white matter structures.

Ocular Delivery of Acetylsalicylic Acid by Repetitive Coulomb-Controlled Iontophoresis

To investigate the potential of transscleral coulomb-controlled iontophoresis (CCI) for repetitive delivery of acetylsalicylic acid (ASA) into the eye, a total of 50 rabbits was included in this study. Fourteen animals received serial CCI treatment. Fourteen animals underwent CCI with either ASA or balanced salt solution (BSS) for at least 6 days at 24- and 48-hour intervals. Eighteen animals received a single CCI application, while 18 animals were injected with 15 mg ASA/kg body weight intravenously. HPLC analysis was performed to determine the levels of salicylic acid (SA) in ocular tissues. Apart from clinical follow-up, 2 rabbits in the ASA and BSS groups were examined by electroretinography, and 2 animals were examined histologically. Though high concentrations of SA were measured, no alterations were observed clinically, histologically and electrophysiologically. Repetitive CCI demonstrated its potential as a topical drug delivery system for ASA into the eye. This transscleral delivery of ASA resulted in significant and sustained intraocular concentrations of SA without side effects. Iontophoresis may be advantageous in clinical administration maintaining therapeutic levels of ASA while avoiding adverse effects associated with the systemic administration of nonsteroidal anti-inflammatory drugs.

Photoreceptor synapses degenerate early in experimental choroidal neovascularization

Severe visual loss in patients with age‐related macular degeneration is associated with the development of choroidal neovascularization (CNV). The pathogenic mechanisms for CNV formation have been extensively investigated, but remarkably little research has addressed the mechanisms for dysfunction of the retina in CNV. Using laser‐induced CNV in mice, we evaluated the mechanisms of retinal dysfunction. At 3 days, 1 week, 2 weeks, and 4 weeks after laser application, retinas under experimental CNV were characterized physiologically (ERG recordings, synaptic uptake of the exocytotic marker FM1‐43, and light‐induced translocation of transducin), histologically, and immunohistochemically. ERG amplitudes were reduced by 20% at 1 week after CNV. Depolarization‐induced FM1‐43 uptake in photoreceptor synapses was selectively reduced by 45% at 1 week after CNV. Although photoreceptor outer segments were shortened by 36%, light adaptation as measured by transducin translocation was mostly preserved. Early in CNV (3 days to 1 week), Muller cells demonstrated induction of c‐fos and pERK expression. Also, the density of macrophage‐like, F4/80 immunoreactive cells increased ∼3‐fold. Minimal photoreceptor death occurred during the first week, and was variable thereafter. At later times in CNV formation (≥2 weeks), expression of photoreceptor synaptic markers was reduced in the outer plexiform layer, indicating loss of photoreceptor synaptic terminals. ERG amplitudes, synaptic uptake of FM1‐43, and the induction of c‐fos and pERK in Muller cells were altered within 1 week of experimental CNV, suggesting that during CNV formation, deficits in retinal function, in particular photoreceptor synaptic function, precede degeneration of photoreceptor terminals and photoreceptor cell death. J. Comp. Neurol. 483:263–277, 2005.

Low-frequency damped electroretinographic wavelets in young asymptomatic patients with dominant retinitis pigmentosa: a new electroretinographic finding.

OBJECTIVE To describe a previously unreported electroretinographic (ERG) pattern in early retinitis pigmentosa (RP). DESIGN Two case reports. PARTICIPANTS Two unrelated young asymptomatic patients with autosomal-dominant retinitis pigmentosa were studied. MAIN OUTCOME MEASURES Clinical findings and ERG responses were assessed. RESULTS No ERG responses were detected scotopically with low-luminance stimuli. With increasingly brighter stimuli, a series of three to five low-frequency damped wavelets developed under both scotopic and photopic conditions. The period of the wavelets was 25 to 37 msec. CONCLUSIONS Low-frequency damped ERG wavelets occur in some young asymptomatic patients with autosomal-dominant RP. The ERG pattern suggests that these wavelets are predominantly cone-generated.

Evidence that a-wave latency of the electroretinogram is determined solely by photoreceptors.

PURPOSE To identify the retinal cells that determine the a-wave latency of rats. METHODS Electroretinograms (ERGs) were recorded from the rod-dominated (0.85% cones) retinas of Long-Evans rats following an intravitreal injection of 1 microL of 40 mM 2-amino-4-phosphonobutyric acid to block the activity of the ON pathway of the second order retinal neurons. ERGs were also recorded following an intraperitoneal injection of sodium iodate to destroy the retinal pigment epithelial (RPE) cells. Damage to a large area of the retina was produced by constant light exposure, and focal damage to the retina was induced by argon laser photocoagulation. The effects of age and anesthesia level on the a-wave latency were also determined. RESULTS Blocking the activity of the ON pathway of the second order retinal neurons did not alter the a-wave latency, and destroying the RPE cells also did not alter the a-wave latency. Damage to a large area of the retina resulted in prolonging the latency but focal retinal damage did not alter the a-wave latency. The a-wave latency was longer in young rat pups but was adult-like by 18 days. The level of anesthesia had no effect on the latency except at very deep stages. CONCLUSIONS The a-wave latency is determined solely by the activity of the photoreceptors. A prolonged latency would indicate that the photoreceptors are damaged over a large area of the retina.

Intranuclear rodlets in the rabbit retina following treatment with MPTP

Retinas from pigmented rabbits treated with N-methyl-4-phenyl-1,2,3,6- tetrahydropyridine (MPTP, a chemical inducer of Parkinsonism in man and monkeys) were studied using light- and electron microscopy. The nuclei of many cells in the inner nuclear layer and the ganglion cell layer of the treated retinas contained crystalloids (intranuclear rodlets) of varying length (0.5-8 microns) which were composed of bundles of 12 nm filaments and which were seen only rarely in untreated retinas. The induced rodlets are oval to round cylinders, 15-20 filaments across (although they are smaller in untreated retinas). Similar intranuclear inclusions have been described under varying conditions in neurons and glia in the central nervous system of several animal species. In rabbits injected acutely with MPTP, most of the affected cells are in the inner nuclear layer in the position of bipolar cells, while in the chronically injected animals, clearly identifiable amacrine cells, and the nuclei of some cells in the ganglion cell layer also contained the inclusions. Evidence is presented that the rodlet-containing cells in the ganglion cell layer include both ganglion cells and displaced amacrine cells. These anatomic findings are relevant to understanding the physiological and biochemical effects of the drug on the retina which we reported previously (Wong, Ishibashi, Tucker and Hamasaki, 1985).

The a-wave latency in control subjects and patients with retinal diseases.

PURPOSE To determine the a-wave latency of the electroretinograms (ERGs) recorded from control subjects and patients with retinal diseases. METHODS The a-wave latency and implicit time (IT) were measured retrospectively from the ERGs of 40 control subjects and 99 patients. The patients included 9 with complete congenital stationary night blindness (cCSNB), 13 with achromatopsia or cone dystrophy, 5 with supernormal and delayed rod ERG syndrome, and 72 with retinitis pigmentosa (RP). To assess whether latency measurements can be obtained reliably by different observers from patients with smaller a-wave amplitudes and noisier baselines, the a-wave latency and IT of the ERG of the right eye of 10 control subjects and 10 patients with RP were measured by three observers. RESULTS The mean a-wave latency measured for the same 10 control ERGs by three observers differed by less than 1 millisecond while the mean IT differed by 1.7 milliseconds. For 10 ERGs from RP patients, the mean for the a-wave latency measured by the three observers differed by less than 2.0 milliseconds and by 1.1 millisecond for the IT. The coefficient of variation varied from 24.8% to 36.7% for the latency and from 11.5% to 16.0% for the IT. The a-wave latencies elicited by the 0-dB stimulus under scotopic and photopic conditions from the 40 control subjects were not statistically different. The a-wave latency in patients with cCSNB did not differ significantly from that in control subjects. The longer a-wave latency in patients with achromatopsia suggested that the rods have a longer latency than cones. The scotopic and photopic a-wave latencies were significantly longer in RP patients. The longer latency in RP patients was not due to smaller a- or b-wave amplitudes. CONCLUSIONS The a-wave latency can be measured as reliably as the IT in control subjects but the reliability is not as good for the latency as for the IT in RP patients. The larger coefficients of variation in RP patients were most likely due to the measurements being made from RP patients at different stages of their disease. Our results suggest that the a-wave latency in control subjects is determined by cones under both scotopic and photopic conditions. The longer a-wave latency in RP patients suggests that the rods and cones are altered over a significant area of the retina.

Alterations of the cat's electroretinogram induced by the lesioning of the indoleamine-accumulating amacrine cells

The neurotoxin 5,7-dihydroxytryptamine (5,7-DHT) was used to destroy the indoleamine-accumulating amacrine cells located in the cats retina. With 100 micrograms of 5,7-DHT, the alterations in the electroretinogram (ERG) were present in all of the treated eyes and the morphological changes were confined to some of the processes in the inner plexiform layer. The ERGs recorded from treated eyes consisted of negative waves at low intensities and depressed b wave amplitude at higher intensities. The duration of the b wave was not altered but the oscillatory potentials were strongly depressed. The changes were probably permanent. The differences in the ERG changes in cats and rabbits were suggested to arise from the differences in signal processing in the inner retina of rabbits and cats.