[A case of fundus albipunctatus with a retinol dehydrogenase 5 gene mutation in a child].

BACKGROUND: We examined a family with fundus albipunctatus in which mutation of the retinol dehydrogenase 5(RDH 5) gene was suspected to be the cause of this disease. CASE: An 8-year-old girl had diffuse multiple white dots in her fundus except for the macula. She had good central vision. The amplitude of her electroretinogram wave was low, but it recovered after three hours of dark adaptation. Dark adaptometry showed an elevated threshold for rod adaptation. No visual field loss was observed. A homozygous missense mutation was found in exon 5 of the RDH 5 gene that substituted histidine for arginine at codon 280(Arg 280 His). Her mother had a normal fundus but was heterozygous for the same mutation. CONCLUSION: A missense mutation of RDH 5(Arg 280 His) was found in a Japanese family with fundus albipunctatus.

Analysis of second-order kernel response components of multifocal electroretinograms elicited from normal subjects.

PURPOSE: It has been reported that the second-order kernel response components of multifocal electroretinograms (mERGs) reflect the electrical activity of the inner retinal layers. In this study, we have investigated whether the amplitudes of the second-order kernel response components correlate with the spatial distribution of human retinal ganglion cells. METHODS: Multifocal electroretinograms were recorded using the Veris III system from 5 healthy subjects with different stimulus and recording parameters. The mERGs were analyzed using the Veris Science software programs. The stimuli consisted of densely arranged arrays of 103, 61, 37 or 19 hexagonal elements. Four minutes were required to record one set of mERG responses using 8 sessions, and 8 minutes using 16 sessions. The second-order kernel response components were extracted and analyzed using the Veris Science program. RESULTS: The signal-to-noise ratio of the first-order kernel response components was improved considerably by the summation of the nine reproducible responses from the same subject but the second-order kernel response components were not. The summation of the nine reproducible responses was insufficient to identify an array of the second-order kernel response components. Both the first- and second-order kernel response components were larger when fewer hexagonal elements were used. There was no significant difference in the individual responses between the 4-minute and the 8-minute recordings. A response density analysis revealed a weak correlation between the amplitude distribution of the second-order kernel response components and the spatial distribution of human retinal ganglion cells. CONCLUSIONS: The distribution of the amplitudes of the second-order kernel response components of the mERGs elicited from normal subjects did not correlate with the distribution of human ganglion cells. This suggests that the theory that second-order kernel response components arise from the activity of retinal ganglion cells should be reconsidered.

Apoptosis after butyrate-induced differentiation in retinoblastoma cell line Y-79.

PURPOSE: To study the fate of Y-79 human retinoblastoma cells after induction of differentiation. METHODS: Y-79 cells were cultured in a synthetic medium and were induced to neuronal differentiation by butyrate treatment. Neurofilaments, p53, and DNA-synthesizing nuclei labeled with 5-bromodeoxyuridine were immunostained, and apoptotic cells were labeled by in situ DNA nick end labeling (TUNEL). We combined these immunostaining and labeling methods to determine whether the cells expressed these markers at the same time. DNA fragmentation and p53 levels were also determined by electrophoresis. RESULTS: Y-79 cells proliferated in the synthetic medium. After butyrate treatment, they extended protrusions and increased neurofilament immunoreactivity. The differentiated features were striking on day 7. Thereafter, differentiated cells decreased and apoptotic cells increased. DNA synthesis was detected in the cells expressing immunoreactivity for neurofilaments or p53. At day 7, most of the cells with p53-positive nuclei were alive and neurofilament-positive. However at day 10, the p53-positive cells were apoptotic and neurofilament-positive apoptotic cells accumulated. CONCLUSIONS: We conclude that the Y-79 cells express p53 and undergo apoptosis after neuronal differentiation. There could be a p53-dependent apoptotic pathway in butyrate-induced differentiated Y-79 cells due to the inability to regulate cell cycling.

The Contribution of Inflammatory Cell Infiltration to Conjunctival Inclusion Cyst Formation.

Purpose: The incidence of inclusion cysts was examined histopathologically in conjunctival disorders where inflammatory cell infiltration was seen in the subepithelial connective tissue.Method: The incidence of inclusion cysts was examined histopathologically in pterygium, vernal conjunctivitis, pyogenic granuloma and pingueculitis. The specimens of pinguecula were used as control.Results: Inclusion cysts were recognized in 5/(55) cases of pterygium, 2/(12) cases of vernal conjunctivitis, 1/(4) cases of pyogenic granuloma, and 1/(2) cases of pingueculitis. On the other hand no inclusion cyst was recognized in 20 cases of pinguecula in which no inflammatory cell infiltration was seen.Conclusions: Inflammatory cell infiltration may contribute to the formation of conjunctival inclusion cysts in pterygium, pingueculitis, vernal conjunctivitis and pyogenic granuloma.

Artifact removal procedure distorts multifocal electroretinogram.

PURPOSE: To study whether the Artifact Removal procedure available for eliminating artifacts in multifocal electroretinograms (mERG) works correctly or not. METHODS: A test response was made using a photo-diode circuit. mERGs were recorded from 3 well-trained normal subjects using the Veris III system, and were then analyzed by the procedure that is included in the Veris Science (Artifact Removal) software program. The stimuli consisted of densely arranged arrays of 103 or 37 hexagonal elements. It took a total of 8 minutes to obtain one mERG record, and 16 sessions were required to complete this record. The first-order as well as the second-order kernel response components were extracted by Veris Science software, and the Artifact Removal procedure was used for both components. RESULTS: The Artifact Removal procedure influenced both the test response on the center element as well as the neighboring traces just around the test response. After the repetitions of the Artifact Removal procedure, the shape of the test response changed considerably. Some of the traces of the second-order kernel response components elicited from a normal subject changed irregularly when the Artifact Removal procedure was repeatedly used. The noise increased at the first iteration of the Artifact Removal procedure. CONCLUSION: This procedure has been considered useful for eliminating artifact distortion in mERG, but should be carefully checked by well-established testing methods before clinical use.

Expression of stem cell factor in pterygium.

PURPOSE: To investigate the possible role of stem cell factor (SCF) in the pathogenesis of pterygium. METHODS: The localization of SCF was examined immunohistochemically in excised tissue from 4 primary pterygia and 5 normal conjunctival specimens. RESULTS: Three of the four pterygia showed strong immunoreactivity of SCF in the subepithelial connective tissue at the cap area. This immunoreactivity was completely blocked by using a primary antibody preincubated with recombinant SCF. The SCF-positive cells were identified as a population of fibroblasts by immunostaining for vimentin and prolyl 4-hydroxylase in adjacent sections. No apparent immunoreactivity of SCF was observed in the subepithelial connective tissues in the head and body of the pterygia and in the normal conjunctiva. CONCLUSION: Stem cell factor is overexpressed in fibroblasts at the cap area of most pterygia.

[The contribution of inflammatory cell infiltration to conjunctival inclusion cyst formation].

PURPOSE: The incidence of inclusion cysts was examined histopathologically in conjunctival disorders where inflammatory cell infiltration was seen in the subepithelial connective tissue. METHOD: The incidence of inclusion cysts was examined histopathologically in pterygium, vernal conjunctivitis, pyogenic granuloma and pingueculitis. The specimens of pinguecula were used as control. RESULTS: Inclusion cysts were recognized in 5/55 cases of pterygium, 2/12 cases of vernal conjunctivitis, 1/4 cases of pyogenic granuloma, and 1/2 cases of pingueculitis. On the other hand no inclusion cyst was recognized in 20 cases of pinguecula in which no inflammatory cell infiltration was seen. CONCLUSIONS: Inflammatory cell infiltration may contribute to the formation of conjunctival inclusion cysts in pterygium, pingueculitis, vernal conjunctivitis and pyogenic granuloma.

Purification and characterization of bovine cone arrestin (cArr).

To elucidate the quenching mechanism of phototransduction in vertebrate cone photoreceptors, a cDNA clone encoding cone specific arrestin (cArr) was isolated from a bovine retinal cDNA library using a human cArr cDNA probe. Affinity-purified anti-peptide antibody specific to cArr was prepared. Immunohistochemical staining displayed specific labeling of cArr in cone photoreceptors and immunoblotting identified a 46 kDa protein band. We purified cArr from bovine retinas by sequential column chromatography using DEAE-cellulose, gel filtration and mono Q columns. Binding studies revealed no binding of cArr to rhodopsin regardless of whether it was bleached and/or phosphorylated. cArr also failed to bind to heparin-Sepharose under conditions which rod arrestin (rArr) bound to the column. The present data suggest that cArr may play a role in the quenching of phototransduction in cone photoreceptors and that its activity therein is different to that of rArr.

A basic investigation of multifocal electroretinogram: reproducibility and effect of luminance.

PURPOSE: To investigate the reproducibility as well as the effect of luminance in multifocal electroretinogram (mERG). METHODS: Multifocal electroretinogram recordings were repeated on different days in 6 normal subjects using the Veris III system. The mean luminance of the monitor displaying the stimuli was randomly varied by five kinds of neutral density (ND) filters. RESULTS: The standard deviation of mERG amplitude from the macular region was approximately 10% of the mean value for each normal subject. Reproducibility largely depended on the condition of the subject and placement of the contact lens electrode. With decreases in the mean luminance of the monitor, the amplitude of mERG decreased exponentially, whereas the peak latency increased linearly. mERGs elicited from a patient with mild cortical cataract resembled the mERGs obtained from the control group using an ND filter between -0.30 and -0.52 log, whereas two patients with typical retinitis pigmentosa showed much lower response densities in mERGs. CONCLUSIONS: It is necessary to pay attention to the reproducibility and the luminance effect to obtain reliable mERGs.

Kerato-epithelin mutation (R 555 Q) in a case of reis-Bucklers corneal dystrophy

Background: Mutations in the kerato-epithelin gene on chromosome 5 q 31 are known to cause four distinct autosomal dominant diseases of the human cornea: Reis-Bucklers, granular, lattice, and Avellino corneal dystrophy. Mutation of arginin to glutamine in codon 555 (R 555 Q) in kerato-epithelin was recently reported in four blood-related patients with Reis-Bucklers corneal dystrophy.Case: A 42-year-old female has had photophobia with decreasing vison since the age of 20 years. Her corrected visual acuity was 0.5 in both eyes. She showed subepithelial opacities in both corneas characteristic of Reis-Bucklers corneal dystrophy.Method: The DNA was extracted from leukocytes according to standard protocols. The keratoepithelin gene was examined for a mutation by the polymerase chain reaction and direct sequencing.Findings: We identified kerato-epithelin mutation R 555 Q. The patient's two children and 50 controls did not show missense mutation.Conclusion: Kerato-epithelin mutation R 555 Q was present in a Japanese patient with Reis-Bucklers corneal dystrophy.

[Kerato-epithelin mutation (R 555 Q) in a case of Reis-Bücklers corneal dystrophy].

BACKGROUND: Mutations in the kerato-epithelin gene on chromosome 5 q 31 are known to cause four distinct autosomal dominant diseases of the human cornea: Reis-Bücklers, granular, lattice, and Avellino corneal dystrophy. Mutation of arginin to glutamine in codon 555 (R 555 Q) in kerato-epithelin was recently reported in four blood-related patients with Reis-Bücklers corneal dystrophy. CASE: A 42 year-old female has had photophobia with decreasing vision since the age of 20 years. Her corrected visual acuity was 0.5 in both eyes. She showed subepithelial opacities in both corneas characteristic of Reis-Bücklers corneal dystrophy. METHOD: The DNA was extracted from leukocytes according to standard protocols. The keratoepithelin gene was examined for a mutation by the polymerase chain reaction (PCR) and direct sequencing. FINDINGS: We identified kerato-epithelin mutation R 555 Q. The patient's two children and 50 controls did not show missense mutation. CONCLUSION: Kerato-epithelin mutation R 555 Q was present in a Japanese patient with Reis-Bücklers corneal dystrophy.

Effects of fluorescent light on growth of bovine retinal pigment epithelial cells in vitro incubated with linoleic acid or linoleic acid hydroperoxide.

Light-induced peroxidation of polyunsaturated fatty acids (PUFA) may generate lipid hydroperoxides, which may have toxic effects on retinal pigment epithelial (RPE) cells in vitro. We investigated the effects of cool-white fluorescent light on the RPE cells incubated with linoleic acids (LA) or linoleic acid hydroperoxides (LHP) and the influence of antioxidative enzymes. We measured the bovine RPE cell number after exposure to fluorescent light (610 and 1,200 lux) in the presence of LA or LHP. Furthermore, the effects of superoxide dismutase (SOD) and catalase on LA- or LHP-treated RPE cells were also examined. Both LA and LHP treatment increased RPE cell number under weak illumination (610 lux), but dose-dependently decreased the number of cells exposed to strong illumination (1,200 lux). With exposure to strong illumination, LA caused a greater reduction in RPE cell number than LHP. Multiple linear regression analysis showed that the number of RPE cells was significantly decreased in a manner dependent on the interactions of the illuminance of light and the concentrations of LA or LHP. The antioxidative enzymes significantly ameliorated the damage to RPE cells from LA or LHP and exposure to light. Therefore, the exposure to fluorescent light augmented the cytotoxic effects of LA and LHP on RPE cells, and this effect is likely to be mediated by reactive oxygen species.

Mast cells in pterygium: number and phenotype.

PURPOSE: To investigate the pathogenesis of pterygium. METHODS: The number and phenotype of mast cells were examined in excised tissue from 35 pterygia patients and compared with those in normal conjunctival specimens obtained during cataract or other intraocular surgery. RESULTS: Toluidine blue staining showed that the mean number of mast cells in the pterygia specimens was twice as high as that in the normal conjunctival tissues. Immunohistochemistry with a primary antibody to tryptase, specific for mast cells, also revealed a twofold increase in the mast cell number in the pterygia specimens compared with the normal conjunctival tissues. In the pterygia, more than 94% of the tryptase-positive mast cells were found to express chymase and c-kit. Almost all mast cells in the pterygia were tryptase-positive, chymase-positive mast cells (MC(TC)S). There was no phenotypic difference between the mast cells in the pterygia and those in the normal conjunctival tissues. CONCLUSIONS: The MC(TC)S appear not to be immune system-related and to have functions in angiogenesis and tissue remodeling. The increase in the number of mast cells caused by nonallergic stimulation may contribute to the pathogenesis of pterygium.

The localization of glutathione peroxidase in the photoreceptor cells and the retinal pigment epithelial cells of Wistar and Royal College of Surgeons dystrophic rats.

INTRODUCTION: If degenerating photoreceptor outer segments not phagocytized by RPE cells in the retina of Royal College Surgeons (RCS) rats were to undergo peroxidation, the distribution of glutathione peroxidase (GSH-PO) in the mitochondria or cytoplasm of the retina might be altered. We evaluated the immunocytochemical localization of GSH-PO to identify subcellular organelles in sections of the retinas of RCS rats. METHODS: Immunoblot analysis confirmed the presence of GSH-PO molecules in the retinas of RCS and Wistar rats aged 3 weeks. Sections were reacted with the F(ab) fragment of anti-rat alphaGSH-PO and then examined by laser scanning microscopy (LSM) and transmission electron microscopy (TEM). RESULTS: The size of the GSH-PO molecule in the retina was about 21 KD in the mitochondria and 23 KD in the cytosol in both strains of rats. LSM revealed fluorescent granules in the photoreceptor inner segments of the Wistar rats, and immunohistochemical TEM revealed GSH-PO in the mitochondria of their photoreceptor inner segments and retinal pigment epithelial (RPE) cells. In the RCS rats, the degenerating photoreceptor outer segments were clearly seen to be positive for anti-GSH-PO by conventional light microscopy (CLM). However, the photoreceptor inner segments of the RCS rats were negative for staining with anti-GSH-PO by LSM, and no GSH-PO could be detected in the mitochondria of the photoreceptor inner segments or RPE cells by immuno-TEM. CONCLUSION: Degeneration of the photoreceptor outer segments induced mitochondrial damage in the photoreceptor inner segments, and as a result GSH-PO shifted from the photoreceptor inner segments to the degenerating outer segments.

[Histopathological observations on bullous keratopathy after argon-laser iridotomy].

PURPOSE: To elucidate histopathological mechanism of bullous keratopathy after argon-laser iridotomy (ALI). CASE AND METHOD: The patient was a 60-year-old female who underwent penetrating keratoplasty because of bullous keratopathy after 2 years and 2 months of ALI. The corneal specimen was fixed with a mixture of 2.5% formalin and 1.0% glutaraldehyde, and examined under light and electron microscopes. FINDING AND CONCLUSION: Laser-damaged endothelium produced a large amount of basement membrane-like material beneath Descemet's membrane. At the next stage, the severely damaged endothelium lost its organellae and cell membranes, and fell off. The surrounding healthy endothelium migrated into the damaged area and produced a small amount of material like basement membrane which covered Descemet's membrane. After that, stromal swelling, decrease of keratocytes, breaking and disappearance of Bowman's membrane, epithelial edema, connective tissue accumulation beneath basal cells, and epithelial detachment occurred in the laser-damaged area.

[Neuronal cell death mechanism in glaucomatous optic neuropathy].

Apoptosis of retinal ganglion cells has been observed in experimental glaucomatous models and human glaucomatous eyes in recent pathological studies. It seems that retinal ganglion cells in glaucomatous optic neuropathy die by a process similar to programmed cell death. Deprivation of neurotrophic factors, ischemia, chronic elevation of glutamate, and disorganized NO metabolism are suspected to be factors affecting neuronal loss in glaucoma, and most of these factors are known to activate the mechanism of cellular suicidal death. One of the common switches for the death mechanism seems to be in the mitochondria and to be controlled by Bcl-2 family proteins. A major goal of neuronal cell death research in glaucomatous neuropathy is to identify its molecular components and mechanisms of regulation. This information will lead to therapeutic agents that can modulate the cell death process in the treatment of glaucomatous neuropathy.

[Detection of parafoveal scotoma by multifocal electroretinograms].

We investigated the relation between multifocal electroretinograms (M-ERGs) and artificial parafoveal scotoma. M-ERGs were recorded from normal subjects using a circular piece of black paper attached to a monitor. Lower response density around the 10 to 15 degree parafovea region was not observed up to 3 degree scotoma (visual angle), but was detected above 5 degree scotoma in field topography of M-ERGs. The shape of the scotoma in field topography was not circular but somewhat oval. The results from two cases of parafoveal retinal degeneration were in good accordance with this basic study in normal subjects. We proved that detection of parafoveal scotoma by M-ERG is limited in comparison with the results obtained by automated static perimetry.

Visual function and gene analysis in a family with Oguchi's disease.

A family with 1 case of retinitis pigmentosa (III-1) and 2 cases of Oguchi's disease (III-2, 3) was examined in terms of electrophysiology as well as molecular biology. The proband (III-3), a 42-year-old female, and 2 older brothers (III-1, 2, aged 52 and 45 years) and 2 unaffected members in the same family participated in this study. Corrected visual acuities of the individuals with Oguchi's disease (III-2, 3) were 1.2. On funduscopy, blood vessels stood out in relief against a metallic-appearing background and a Mizuo-Nakamura phenomenon was evident. Full-field electroretinograms (ERGs) recorded from the proband were indicative of rod dystrophy, but results of other electrophysiological examinations (multifocal ERG, pattern ERG and visual-evoked cortical potential recordings) were within normal limits. Patient III-1 had corrected visual acuities of RE 20 cm/m.m. and LE 30 cm/n.d., severe chorioretinal atrophy in both fundi, and full-field ERG revealed rod-cone dystrophy. Mutation of the arrestin gene (1147de1A) was detected in all 3 patients. Visual function in each patient coincides with that of retinitis pigmentosa or Oguchi's disease, respectively.

Automatic static threshold perimetry is useful for estimating the effects of laser photocoagulation on diabetic maculopathy.

Focal photocoagulation was performed on 18 eyes in 18 diabetes patients; a visual acuity test, fluorescein angiography and automated static threshold perimetry by Octopus automated static perimetry (program 31) were conducted before and 1, 3 and 6 months after krypton laser photocoagulation. Central field sensitivity and total loss were measured by automated static threshold perimetry. Group A (13 eyes) had a total loss of less than 150 dB. In these subjects, mild fluorescein leakage was detected within the area of the vascular arcade. In group B (5 eyes) with a total loss of greater than 150 dB, diffuse fluorescein leakage was also detected outside the vascular arcade. The central field sensitivity was reduced in 2 eyes of group B. The total loss worsened in 8 eyes of group A (61.5%) and 5 eyes of group B (100%). Inspection of photographs gave the impression that the degree of fluorescein leakage and the area of the avascular zone on fluorescein angiography accorded with total loss, thus suggesting that total loss reflects visual functions better than visual acuity or central field sensitivity. Therefore, automated static threshold perimetry appears to be a useful method for estimating visual functions after photocoagulation in diabetic maculopathy patients.

[Pterygium and mast cells--expression of basic fibroblast growth factor increase in mast cells of the pterygium].

We examined the expression of basic fibroblast growth factor (bFGF) protein immunohistochemically, and bFGF specific messenger RNA (bFGF-mRNA) by in situ hybridization in the excised tissue of 5 cases of pterygium and 4 cases of normal conjunctiva. Immunohistochemical staining for bFGF showed strong positive staining in metachromatic mast cells stained with toluidine blue in the pterygium and in normal conjunctival specimens. The mean metachromatic mast cell count in pterygium specimens was increased significantly when compared with normal conjunctiva. In mast cells, bFGF positive rate was 84% in pterygium specimens, and 69% in normal conjunctival specimens. In situ hybridization indicated that the bFGFmRNA is located in most mast cells in pterygium specimens, but in only a few mast cells in normal conjunctival specimens. These results suggest that increased bFGF protein produced and stored by mast cells in the pterygium may contribute to its progression.