Shigekuni Okisaka

Apoptosis in retinal ganglion cell decrease in human glaucomatous eyes

Hematoxylin-eosin staining, the TUNEL method for in situ detection of the intranuclear DNA fragmentation, which indicates apoptosis, and electron microscopy were used to study the morphologic changes in specimens from the eyes of 8 patients with secondary glaucoma and 2 normal control eyes to evaluate our hypothesis that apoptosis causes a decrease in retinal ganglion cells in human glaucomatous eyes. The TUNEL method permits identification of intranuclear DNA fragmentation. Apoptosis was found in the ganglion cells of 2 glaucomatous eyes with recent sight loss, and in the ganglion cells of a control eye from a 95-year-old subject, taken at autopsy. Results of our study indicate that a decrease in retinal ganglion cells in glaucomatous eyes is caused by apoptosis. In addition, apoptosis resulting from aging must be considered in order to understand the reduction of retinal ganglion cells in the glaucomatous eyes of elderly patients.

Mechanism of intraocular pressure decrease after contact transscleral continuous-wave Nd:YAG laser cyclophotocoagulation.

Twenty-two eyes of 11 cynomolgus monkeys were subjected to contact transscleral cyclophotocoagulation with a continuous-wave Nd:YAG laser. The right eye of each monkey was coagulated at the pars plicata region by the contact probe placed 1.0 mm from the limbus, while the left eye of each monkey was coagulated at the pars plana region by the contact probe placed 3.0 mm from the limbus. Physiological and morphological studies were carried out up to 6 months after the treatment. The postoperative intraocular pressure showed a significant decrease within 1 week, corresponding to the inflammation of the anterior chamber. A gradual increase of the intraocular pressure occurred from the 2nd week on and returned to the preoperative value 8 weeks after pars plicata coagulation. The pars plana coagulation group maintained the intraocular pressure lower than the preoperative value until the end of the observation period. Histopathological examinations were carried out by the use of tracer particle perfusion into the anterior chamber. The pathologic features of pars plicata coagulation were necrosis, followed by atrophy of the ciliary process. The tracer particles accumulated at the anterior portion of the space between the bundles of ciliary muscle. The pathologic features of pars plana coagulation were necrosis followed by extension of proliferative tissue into the vitreous. The surrounding extracellular space of the stroma was enlarged, and the ciliary muscles were separated from the sclera. The tracer particles accumulated at the enlarged extracellular space of the stroma and the opened suprachoroidal space. These results suggest that the decrease of the intraocular pressure after pars plicata cyclocoagulation resulted from the reduction of aqueous secretion, whereas that after pars plana cyclocoagulation resulted from enhancement of the uveoscleral outflow through the enlarged extracellular space from the anterior chamber into the suprachoroidal space.

Autoantibody Against Neuron-Specific Enolase Found in Glaucoma Patients Causes Retinal Dysfunction In Vivo

PURPOSE In our recent paper, we have reported the presence of serum autoantibody against neuron-specific enolase (NSE) in patients with glaucoma. The purpose of the present study was to investigate further the pathological effects of anti-NSE antibody on retina by comparing them with the effects induced by N-methyl-D-aspartate (NMDA). METHODS Either a glaucoma patients serum or purified anti-NSE antibody, or 10-40 mM NMDA was intravitreously administered into Lewis rat eyes, and electrophysiological, histopathological, and biochemical evaluations were performed. In addition, the neuroprotective effects of anti-glaucoma drugs, such as timolol, betaxolol, nipradilol, and isopropyl unoprostone, and a calcium antagonist were also studied using these animal models. RESULTS Electron microscopy revealed that intravitreal administration of a glaucoma patients serum, which immunoreacted with retinal 50 kDa in Western blot analysis, and purified anti-NSE antibody induced retinal ganglion cell apoptosis in rat eyes. Functionally, these eyes showed a significant decrease in electroretinogram (ERG) responses and a remarkable decrease in rhodopsin phosphorylation reaction. These changes were comparable to the effects observed after the intravitreal administration of 20 mM NMDA. Co-administration of nipradilol, an alpha- and beta-blocker, with anti-NSE antibody or 20 mM NMDA caused marked recovery of the affected ERG responses within 2 weeks. In contrast, administration of timolol or betaxolol showed no recovery effect on the ERG responses. Among these drugs, only betaxolol showed a recovery effect on NMDA-induced decrease of rhodopsin phosphorylation. Nilvadipine functioned beneficially on both impaired ERG and rhodopsin phosphorylation reactions observed in rat eyes injected intravitreously with anti-NSE antibody or NMDA. These effects of nilvadipine were not changed by the addition of endothelin-1. In contrast, isopropyl unoprostone had no effect on these functions. CONCLUSION These observations suggest that serum autoantibody against NSE found in some patients with glaucoma induces retinal dysfunction in vivo, similarly to NMDA.

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.

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.

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.

Comparative effects of linoleic acid and linoleic acid hydroperoxide on growth and morphology of bovine retinal pigment epithelial cells in vitro

PURPOSE Outer segments of the photoreceptor rods that are phagocytized by the retinal pigment epithelial (RPE) cells contain a high proportion of polyunsaturated fatty acids (PUFA). PUFA are susceptible to lipid peroxidation. We hypothesized that the resulting peroxides could injure RPE cells leading to retinal degeneration. Accordingly, we compared the effects of linoleic acid (LA) and its hydroperoxide (LHP) on the growth and morphology of RPE cells using laser scanning microscopy and transmission microscopy. METHODS We counted the number of RPE cells after incubation for 24 and 48 hrs with concentrations of LA or LHP of 0.035, 0.175, and 0.35 mM. To observe the actin filaments, cultured RPE cells were stained with rhodamine phalloidin. The cells were prefixed with 2% glutaraldehyde and postfixed in 1% osmium tetroxide. Specimens were embedded in Epon 812 after dehydration, and the ultrathin sections were doubly stained with 2% uranyl acetate and 2% lead acetate for examination by transmission electron microscopy. RESULTS Exposure to LA or LHP produced dose-dependent damage to RPE cells with a significantly greater effects of LHP than LA. After incubation for 24 hrs with 0.35 mM LA, the number of vacuoles in RPE cells exceeded that observed in control RPE cells by 365 nm laser microscopy. Exposure to 0.35 mM LHP for 24 hrs produced a pycnotic nucleus, with diffuse and granular autofluorescences observed in and around it. Exposure of RPE cells to 0.35 mM LA for 24 hrs showed that the LA incorporated into the lysosomes was digested and released extracellularly from lysosomes via exocytotic vesicles. However, such exposure to LHP damaged the RPE cells, including the membranes in the pinocytotic vesicles. The packed membranes resembled myelin. CONCLUSIONS While the LA incorporated into the lysosomes was released extracellularly, LHP persisted in the RPE cells, being observed as autofluorescent lipofuscin-like materials. LHP was cytotoxic, and caused damage to the membranes of pinocytotic vesicles and lysosomes.

Relationship between visual field defect and multifocal electroretinogram.

We investigated the effect of artificial parafoveal scotomata on the multifocal electroretinogram (M-ERG). M-ERGs were recorded from normal subjects using a monitor with several different sizes of black paper attached. A lower response density area around the 10 to 15 degree parafoveal region was not recognized for scotomata up to 3 degrees but was observed in scotomata above 5 degrees (visual angle) in the field topography of M-ERG. The shape of the scotomata was not circular but somewhat oval. The results from two cases of parafoveal retinal degeneration accorded well with this basic study.

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 cases, but no mutation was observed for the rhodopsin gene. A homozygous deletion 1147 (1147de1A) in codon 309 of the arrestin gene was commonly observed 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.