METTL23 mutation alters histone H3R17 methylation in normal-tension glaucoma.

Normal-tension glaucoma (NTG) is a heterogeneous disease characterized by retinal ganglion cell (RGC) death leading to cupping of the optic nerve head and visual field loss at normal intraocular pressure (IOP). The pathogenesis of NTG remains unclear. Here, we describe a single nucleotide mutation in exon 2 of the methyltransferase-like 23 (METTL23) gene identified in 3 generations of a Japanese family with NTG. This mutation caused METTL23 mRNA aberrant splicing, which abolished normal protein production and altered subcellular localization. Mettl23-knock-in (Mettl23+/G and Mettl23G/G) and -knockout (Mettl23+/- and Mettl23-/-) mice developed a glaucoma phenotype without elevated IOP. METTL23 is a histone arginine methyltransferase expressed in murine and macaque RGCs. However, the novel mutation reduced METTL23 expression in RGCs of Mettl23G/G mice, which recapitulated both clinical and biological phenotypes. Moreover, our findings demonstrated that METTL23 catalyzed the dimethylation of H3R17 in the retina and was required for the transcription of pS2, an estrogen receptor α target gene that was critical for RGC homeostasis through the negative regulation of NF-κB-mediated TNF-α and IL-1ß feedback. These findings suggest an etiologic role of METTL23 in NTG with tissue-specific pathology.

Establishment of an experimental ferret ocular hypertension model for the analysis of central visual pathway damage.

Glaucoma optic neuropathy (GON) is a condition where pathogenic intraocular pressure (IOP) results in axonal damage following retinal ganglion cell (RGC) death, and further results in secondary damage of the lateral geniculate nucleus (LGN). Therapeutic targets for glaucoma thus focus on both the LGN and RGC. However, the temporal and spatial patterns of degeneration and the mechanism of LGN damage have not been fully elucidated. Suitable and convenient ocular hypertension (OH) animal models with binocular vision comparable to that of monkeys are strongly needed. The ferret is relatively small mammal with binocular vision like humans - here we report on its suitability for investigating LGN. We developed a new method to elevate IOP by injection of cultured conjunctival cells into the anterior chamber to obstruct aqueous outflow. Histologically, cultured conjunctival cells successfully proliferated to occlude the angle, and IOP was elevated for 13 weeks after injection. Macroscopically, the size of the eye gradually expanded. Subsequent enlargement of optic nerve head cupping and atrophic damage of LGN projected from the OH eye were clearly observed by anterograde staining with cholera toxin B. We believe the ferret may be a promising OH model to investigate secondary degeneration of central nervous system including LGN.

Brimonidine is neuroprotective against glutamate-induced neurotoxicity, oxidative stress, and hypoxia in purified rat retinal ganglion cells.

PURPOSE: To investigate the neuroprotective effect of alpha2-adrenergic agonist brimonidine in the presence of glutamate-induced neurotoxicity, oxidative stress, and hypoxia on in vitro cultures of purified rat retinal ganglion cells (RGCs). METHODS: Purified RGC cultures were obtained from retinas of 6-8-day old Wistar rats, following a two-step immunopanning procedure. After 72 h of cultivation, the neuroprotective effect of brimonidine (0.01 microM, 0.1 microM, and 1 microM) was investigated by culturing the RGCs under glutamate, oxidative, and hypoxic stress for a further 72 h, 24 h, and 12 h, respectively. Glutamate neurotoxicity was induced by adding glutamate (25 microM), while oxidative stress was induced by substituting the culture medium with B27 supplement without antioxidants, and hypoxia was induced by cultivation in a controlled-atmosphere incubator with oxygen levels 5% of the normal partial pressure. The RGC viability under each stress condition normalized to that under normal condition was evaluated as live cell percentage based on a total of 7-8 full repeated experiments. RESULTS: The cell survival percentages of cultures exposed to glutamate, oxidative, and hypoxic stress were 58.2%, 59.3%, and 53.2%, respectively. Brimonidine dose dependently increased RGC survival in the presence of glutamate (80.6% at 1 microM), oxidative (79.8% at 1 microM), and hypoxic (72.3 and 77.4% at 0.1 and 1 microM, respectively) stress. In the presence of alpha2-adrenergic antagonist yohimbine (10 microM), brimonidine (1 microM) showed no protective effects on RGC viability. CONCLUSIONS: At a concentration of 0.1 microM or higher, brimonidine increased survival of purified rat RGCs in the presence of glutamate neurotoxicity, oxidative stress, and hypoxia. The neuroprotective effect of brimonidine is mediated via alpha2-adrenergic receptors at the RGC level.

Morphological changes in the visual pathway induced by experimental glaucoma in Japanese monkeys.

Glaucoma, an optic neuropathy, is the leading cause of world blindness. In this condition, the damage extends from the retina to the visual center in the brain, although the primary region of damage is thought to be the optic nerve head (ONH), with the lateral geniculate nucleus (LGN) being secondarily affected. We investigated time-dependent alterations in the ONH, the optic nerve (ON), and the LGN after intraocular pressure (IOP) elevation in Japanese monkeys (a species more similar to humans than other macaque species). Nine Japanese monkeys, each with an experimental glaucomatous left eye, and two naive monkeys were studied. Ocular-testing sessions (including IOP measurement and fundus photography) were held weekly. Eyes and brains were enucleated at 2-48 weeks after IOP elevation, and alterations in ONs and LGN were evaluated. The IOP of the treated eyes was monitored periodically and found to be elevated continuously throughout the observation period in each monkey. The ONH of the glaucomatous eyes exhibited time-dependent deep cupping and thinning of the rim area from 2 weeks after the IOP elevation. Loss of axons and a decrease in the area of ON were first observed at 4 and 28 weeks, respectively. Neuronal loss was first observed at 2 weeks in layers 1 and 2 of LGN [magnocellular (M)-layer] and at 12 weeks in layers 3-6 of LGN [parvocellular (P)-layer]. Neuronal shrinkage was first observed at 2 weeks in all layers in LGN. These findings indicate that in Japanese monkeys, damage to neurons in LGN can be detected in the early phase (first few weeks) after an IOP elevation, as can damage to ONH.

A placebo-controlled 3-year study of a calcium blocker on visual field and ocular circulation in glaucoma with low-normal pressure.

PURPOSE: To study the 3-year effect of oral nilvadipine, a calcium antagonist, on visual field performance and ocular circulation in open-angle glaucoma (OAG) with low-normal intraocular pressure (IOP). DESIGN: A randomized, placebo-controlled, double-masked, single-center trial. PARTICIPANTS: Patients with OAG who were younger than 65 years and had untreated IOP consistently of 16 mmHg or less. INTERVENTION: Oral nilvadipine (2 mg twice daily) or placebo was assigned randomly to patients fulfilling the criteria by the minimization method of balancing the groups according to age, refraction, and the mean deviation (MD) value (Humphrey Perimeter 30-2 SITA Standard Program; Humphrey Instruments, Inc., San Leandro, CA) of the eye with less negative MD. No topical ocular hypotensive drugs were prescribed. Visual field testing was performed every 3 months; fundus examination and IOP, blood pressure, and pulse rate measurements were carried out every month; and quantitative indexes of circulation in the optic disc rim (NB(ONH)) and choroid in the foveal area (NB(fovea)) were determined using the laser speckle method at 0, 3, 6, 12, 18, 24, 30, and 36 months. MAIN OUTCOME MEASURES: The time courses of MD, NB(ONH), and NB(fovea) in the eye with less negative MD. RESULTS: Thirty-three patients were enrolled; 17 were assigned to nilvadipine and 16 were assigned to placebo; 13 in each group completed the study. No significant intergroup difference was seen in age, refraction, or baseline values of any of the parameters. During the 3-year period, the IOP averaged 12.6 mmHg in the nilvadipine group and 12.8 mmHg in the placebo group (P>0.1), and no significant change from baseline or intergroup difference was seen in blood pressure or pulse rate. The estimated slope of change in the MD was less negative in the nilvadipine than in the placebo group (-0.01 vs. -0.27 decibels/year; P = 0.040). The NB(ONH) and NB(fovea) values remained increased compared with baseline for the study period by approximately 30% to 40% only in the nilvadipine group, and the intergroup difference was significant (P = 0.003 for NB(ONH) and P = 0.007 for NB(fovea)). CONCLUSIONS: Nilvadipine (2 mg twice daily) slightly slowed the visual field progression and maintained the optic disc rim, and the posterior choroidal circulation increased over 3 years in patients with OAG with low-normal IOP.

Optic disc and peripapillary morphology in unilateral nonarteritic anterior ischemic optic neuropathy and age- and refraction-matched normals.

PURPOSE: To compare optic disc morphologic features and peripapillary retinal nerve fiber layer (RNFL) thickness between the unaffected eyes of patients with unilateral nonarteritic anterior ischemic optic neuropathy (NAION) and their affected eyes and the eyes of age- and refraction-matched normal control subjects. DESIGN: Cross-sectional comparative study. PARTICIPANTS: Thirty-one patients with unilateral NAION and 62 age- and refraction-matched normal control subjects. METHODS: Optic disc morphologic features and peripapillary RNFL thickness were evaluated in both eyes of patients with unilateral NAION and in one randomly chosen eye of the normal control subjects. MAIN OUTCOME MEASURES: Optic disc and cup parameters were measured using the Heidelberg Retina Tomograph II (Heidelberg Engineering GmbH, Dossenheim, Germany), and RNFL thickness was measured by scanning laser polarimetry with variable corneal compensation (GDx VCC; Carl Zeiss Meditec, Dublin, CA). RESULTS: There was no significant difference in the disc area between the NAION affected eyes and the unaffected fellow eyes. The cup area, cup-to-disc area ratio, cup volume, and cup shape measure were greater, whereas the peripapillary RNFL thickness was smaller in the former than the latter (P = 0.001 to approximately 0.043). When the unaffected eyes of patients with NAION and the age- and refraction-matched normal control eyes were compared, the disc area, cup area, cup-to-disc area ratio, cup volume, mean cup depth, and cup shape measure were smaller in the former (P = 0.0006 to approximately 0.03); there was no significant difference in the RNFL thickness between the two (P>0.06). CONCLUSIONS: A comparison of the eyes with NAION and the fellow eyes indicated that the cup was slightly larger in the former than in the latter, suggesting the acquired enlargement of the cupping after NAION develops. A comparison of the unaffected fellow eyes in patients with NAION and the age- and refraction-matched normal control eyes suggested that a smaller disc area and smaller cupping were predisposing risk factors for the development of NAION.

Optic disc topography and peripapillary retinal nerve fiber layer thickness in nonarteritic ischemic optic neuropathy and open-angle glaucoma.

OBJECTIVE: To evaluate the results of scanning laser tomography and scanning laser polarimetry (SLP) and the correlations with visual field damage (VFD) in eyes with nonarteritic ischemic optic neuropathy (n-AION) compared with eyes with open-angle glaucoma (OAG). DESIGN: Cross-sectional study. PARTICIPANTS: Thirty-three eyes of 33 patients with n-AION and 33 eyes with OAG whose age and VFD evaluated with the Humphrey field analyzer were matched to those of the n-AION eyes. MAIN OUTCOME MEASURES: The parameters of optic disc topography obtained with the Heidelberg Retina Tomograph II (HRT II) and retinal nerve fiber layer (RNFL) thickness with GDx with variable corneal compensation and the correlation to VFD. RESULTS: The cup area, cup-to-disc area ratio, and mean cup depth were significantly smaller, and the cup shape measure more negative, in the n-AION eyes than in the OAG eyes (P<0.001), whereas rim area was significantly greater (P<0.001). Multivariate analyses showed that none of disc area, rim area, and mean cup depth in the n-AION eyes and only rim area (P = 0.029) in the OAG eyes was significantly associated with mean deviation (MD). Ellipse average of RNFL thickness significantly correlated with MD in the n-AION eyes (P = 0.045) and in the OAG eyes (P = 0.022). CONCLUSIONS: Disc topography of eyes with n-AION was quantitatively characterized by small and shallow cupping and a relatively large rim area compared to eyes with OAG matched for age and VFD. In eyes with n-AION, significant correlation with VFD was found only for the RNFL thickness evaluated with SLP but not for the HRT II parameters.

Frequency doubling technology and scanning laser tomography in eyes with generalized enlargement of optic disc cupping.

PURPOSE: To characterize functional and structural changes in eyes with generalized enlargement of optic disc cupping (vertical cup/disc ratio > or = 0.8), normal intraocular pressure, normal standard achromatic automated perimetry (SAP) results, and no other ophthalmoscopic findings suggesting glaucoma (large C/D eyes) using frequency doubling technology (FDT) and the Heidelberg Retina Tomograph (HRT). METHODS: This comparative observational case series included 30 large C/D eyes (30 subjects), 17 eyes (17 patients) with early-stage normal tension glaucoma with generalized enlargement of optic disc cupping (NTG eyes), and 25 eyes from 25 normal subjects (normal eyes). Results with Humphrey 30-2, FDT N-30 threshold programs, and HRT were compared among these groups. Large C/D eyes were subdivided into FDT-normal and -abnormal eyes according to the predetermined criteria and HRT parameters were compared among them. RESULTS: No significant difference was seen in HRT parameters between the large C/D and NTG eyes. In the large C/D eyes, FDT mean deviation was lower than in the normal eyes and higher than in the NTG eyes, whereas FDT pattern standard deviation was smaller than in the NTG eyes (P = 0.02-0.03). Among HRT parameters, only cup shape measure (CSM) showed significant negative correlation with FDT mean deviation in the large C/D eyes. Between FDT-normal and -abnormal subgroups, only CSM showed significant difference (P < 0.01). CONCLUSION: Frequency doubling technology showed abnormalities in large C/D eyes. Only CSM showed significant correlation with FDT result and difference between those with normal and abnormal FDT results. In management of large C/D eyes, FDT and CSM will be useful to detect functional and structural change.

Long-term outcome of iron-endocytosing cultured corneal endothelial cell transplantation with magnetic attraction.

The long-term efficacy and safety of transplanting iron-endocytosing cultured corneal endothelial cells (CECs) with magnetic attraction were evaluated. Rabbit corneas were subjected to cryo-injury to detach CECs. Cultured rabbit CECs (RCEC) were exposed to spherical iron powder and then injected into the anterior chamber, after which a neogium magnet was fixed on the eyelid for 24 hr to attract the cells to Descemet's membrane (RCEC-iron group, n=4). An RCEC group (cryo-injury and injection of normal cultured RCEC, n=4) and a Cryo group (cryo-injury without injection of RCEC, n=4) served as controls. Intraocular pressure was measured for 12 months after surgery. Corneal findings on slit lamp biomicroscopy, RCEC density, the electro-retinogram (ERG), and residual iron in the ocular tissues were evaluated at final assessment. Intraocular pressure did not increase in any group throughout 12 months of observation. At the final assessment, the average corneal edema score of the RCEC-iron group was significantly lower than that of the RCEC or Cryo groups (p=0.021). The average CEC density of the RCEC-iron group was 2581+/-230 cells mm(-2) (mean+/-SD), whereas no CECs were observed on the inner surface of the central cornea in the RCEC and Cryo groups. No significant differences of the ERG (a- and b-wave amplitudes, and b-wave/a-wave ratio) were detected among the groups. Iron powder was not detected by Berlin blue staining in the ocular tissues of the RCEC-iron group. Apoptotic cells were not observed in the endothelium by terminal transferase-mediated nick-end labeling. Transplanted iron-endocytosing RCEC remained viable for 12 months after surgery. There were no detectable ocular complications after the transplantation of iron-endocytosing cultured RCEC. Magnetic attachment of iron-endocytosing CECs can be an effective and safe method for corneal endothelial repair.

Correlations between allergen-specific IgE serum levels in patients with allergic conjunctivitis in spring.

PURPOSE: The purpose of this study was to evaluate the relation between various specific class E immunoglobulins (IgE) in serum and allergic conjunctivitis in spring. METHODS: Forty patients with allergic conjunctivitis and fifty healthy volunteers were selected for this trial. Total IgE and specific IgE to twelve inhalant allergens were measured using the CAP system. RESULTS: Specific IgE levels caused by cedar and cypress pollen, house dust, Dermatophagoides pteronyssinus, acarus, and moth allergens were higher in the allergy group than in the control group. No correlation was proven between the specific IgE levels and the total IgE level. The highest positivity rate for a specific allergen in patients with allergic conjunctivitis was 52.5% for cedar pollen, followed by cypress pollen at 37.5%. CONCLUSIONS: These results suggest that cypress pollen was the largest cause of allergic conjunctivitis and that the total IgE level had no correlation with each specific IgE.

Late-onset transconjunctival oozing and point leak of aqueous humor from filtering bleb after trabeculectomy.

PURPOSE: To estimate the prevalence and identify factors related to late-onset transconjunctival aqueous oozing and point leak from functioning blebs after trabeculectomy with 5-fluorouracil (5-FU) or mitomycin C. DESIGN: Cross-sectional study. SETTING: The study took place at the outpatient clinic of the Department of Ophthalmology, University of Tokyo Graduate School of Medicine (Tokyo, Japan). Four hundred three consecutive patients (403 eyes) with functional blebs at least 3 months after previously performed trabeculectomy were examined between December 1997 and February 1998. The Seidel test was performed with extended observation up to 15 seconds. Oozing was identified as transconjunctival aqueous egress without interruption of the conjunctival tissue or aqueous stream on the bleb wall. Logistic regression analyses of oozing and point leak in 331 eyes with an avascular area were performed using independent variables, including age, gender, glaucoma diagnosis, prior incisional surgery, antimetabolite use, combined cataract surgery, postoperative follow-up period, intraocular pressure (IOP), concurrent glaucoma therapy, bleb size, and avascular area size. RESULTS: Of 403 eyes, 48 eyes (11.9%) had oozing and 8 eyes (2.0%) had point leak. Intraocular pressure was significantly lower and an avascular area was more frequent in eyes with oozing or leak than in eyes without (P <.001). Logistic regression analysis revealed that oozing was significantly more common after use of 5-FU than mitomycin C (P =.024), whereas point leak was associated with a larger avascular area (P =.045). CONCLUSIONS: After trabeculectomy with antimetabolites, transconjunctival oozing is much more frequent than point leak. Oozing was significantly associated with the use of 5-FU and point leak with a larger avascular area in the bleb.