Association study of genetic variants on chromosome 7q31 with susceptibility to normal tension glaucoma in a Japanese population.

The caveolin 1 to caveolin 2 (CAV1-CAV2) gene region on chromosome 7q31 has been reported to be associated with susceptibility to primary open angle glaucoma (POAG) and normal tension glaucoma (NTG) in previous studies. We investigated whether genetic variants in the CAV1-CAV2 region are associated with NTG in Japanese patients. Two hundred and ninety-two Japanese patients with NTG and 352 Japanese healthy controls were recruited. We genotyped three single-nucleotide polymorphisms; that is, rs1052990, rs4236601, and rs7795356, in the CAV1-CAV2 gene region and assessed the allelic diversity among cases and controls. The frequency of the minor allele (G) of rs1052990 was significantly decreased in NTG cases compared with controls (P=0.014, OR=0.71), whereas NTG or POAG cases had a significantly higher frequency of the allele than controls in previous studies. Conversely, rs7795356 did not show any significant association with NTG cases, and rs4236601 was monomorphic in the Japanese study population. Our findings did not correspond with previous positive results, suggesting that CAV1-CAV2 variants studied in the present study are not important risk factors for NTG susceptibility in all populations. Further studies are needed to elucidate the possible contribution of the CAV1-CAV2 region to the development of glaucoma.

Investigation of the association between the GLC3A locus and normal tension glaucoma in Japanese patients by microsatellite analysis.

PURPOSE: To investigate whether the GLC3A locus harboring the CYP1B1 gene is associated with normal tension glaucoma (NTG) in Japanese patients. MATERIALS AND METHODS: One hundred forty-two Japanese patients with NTG and 101 Japanese healthy controls were recruited. Patients exhibiting a comparatively early onset were selected as this suggests that genetic factors may show stronger involvement. Genotyping and assessment of allelic diversity was performed on 13 highly polymorphic microsatellite markers in and around the GLC3A locus. RESULTS: There were decreased frequencies of the 444 allele of D2S0416i and the 258 allele of D2S0425i in cases compared to controls (P = 0.022 and P = 0.034, respectively). However, this statistical significance disappeared when corrected (Pc > 0.05). We did not find any significant association between the remaining 11 microsatellite markers, including D2S177, which may be associated with CYP1B1, and NTG (P > 0.05). CONCLUSIONS: Our study showed no association between the GLCA3 locus and NTG, suggesting that the CYP1B1 gene, which is reportedly involved in a range of glaucoma phenotypes, may not be an associated factor in the pathogenesis of NTG.

Microsatellite analysis of the GLC1B locus on chromosome 2 points to NCK2 as a new candidate gene for normal tension glaucoma.

AIMS: The aim of this study was to investigate the association between normal tension glaucoma and the candidate disease locus glaucoma 1, open angle, B (GLC1B) on chromosome 2. There are many reports describing the results of association or linkage studies for primary open angle glaucoma (POAG), with GLC1B as one of the loci associated with normal or moderately elevated intraocular pressure. However, there are few reports about the association of genes or defined genomic regions with normal tension glaucoma, which is the leading type of glaucoma in Japan. The GLC1B locus is hypothesized to be a causative region for normal tension glaucoma. METHODS: Genomic DNA was extracted from whole blood of normal tension glaucoma (n = 143) and healthy controls (n = 103) of Japanese origin. RESULTS: Fifteen microsatellite markers within and/or near to the GLC1B locus were genotyped, and their association with normal tension glaucoma was analysed. Two markers D2S2264 and D2S176 had significant positive associations. CONCLUSION: The D2S176 marker had the strongest significant association and it is located 24 kb from the nearest gene NCK2, which now becomes an important new candidate gene for future studies of its association with normal tension glaucoma.

Trans-Tenon's retrobulbar triamcinolone acetonide injection for macular oedema related to branch retinal vein occlusion.

BACKGROUND: The aim of the study was to evaluate the safety and effectiveness of trans-Tenon's retrobulbar triamcinolone acetonide (TA) injection for macular oedema associated with branch retinal vein occlusion (BRVO). METHODS: We reviewed the medical records of 50 consecutive patients with macular oedema associated with BRVO who were treated with trans-Tenon's retrobulbar TA injection (20 mg) as initial treatment for a follow-up period of at least 12 months. Foveal thickness determined by optical coherence tomography, visual acuity, intraocular pressure (IOP) and cataract progression were measured. RESULTS: The mean duration between oedema onset and TA injection was 4.9 months. Foveal thickness decreased significantly at 3 months after injection (p<0.0001). Furthermore, the percentage reduction in foveal thickness in eyes with posterior vitreous detachment (PVD; n = 23) was significantly greater than that without PVD (n = 27, p = 0.003). Improved visual acuity by at least 0.20 log minimum angle of resolution (logMAR) was seen in 22 eyes (44%; 11 eyes with PVD and 11 eyes without PVD). After completion of the 3-month follow-up, 29 eyes (58%) needed additional treatment including TA injections or pars plana vitrectomy (PPV). PPV seemed to be effective for macular oedema resistant to TA. IOP elevation and cataract progression occurred in less than 10% of all patients. CONCLUSIONS: Trans-Tenon's retrobulbar TA injection appeared safe and relatively effective for macular oedema associated with BRVO. In eyes resistant to TA injection, PPV may be effective as an adjunctive treatment.

Upregulated expression of N-syndecan, a transmembrane heparan sulfate proteoglycan, in differentiated neural stem cells.

Adult rat hippocampus-derived neural stem cells are incorporated into neural tissues, and differentiate to neuronal and glial cells. However, the cell surface protein molecules are, to date, undefined. RT-PCR, immunoblotting and immunocytochemistry showed the increased expression of N-syndecan, a transmembrane heparan sulfate proteoglycan, in the neural stem cells after the differentiation induced by retinoic acid. Our data indicate that N-syndecan may be involved in the differentiation of neural stem cells.

Novel cytochrome P4501B1 (CYP1B1) gene mutations in Japanese patients with primary congenital glaucoma.

PURPOSE: To investigate CYP1B1 gene mutations in Japanese patients with primary congenital glaucoma (PCG). METHODS: Sixty-five unrelated Japanese patients with PCG were screened by PCR-single-strand conformational polymorphism (SSCP) analysis followed by direct sequencing. No patients were offspring of consanguineous marriages, a common occurrence among patients in previous reports. PCG haplotypes were constructed with intragenic polymorphisms in affected individuals. Three-dimensional atomic structures of human CYP1B1 and four mutant CYP1B1 sequences representing missense mutations were assembled using homology modeling and were regularized by an energy-minimization procedure. RESULTS: Eleven novel mutations, including seven definite and four probable mutations, were detected in 13 (20%) of the 65 unrelated patients. Of the seven definite mutations, three were predicted to truncate the CYP1B1 open reading frame. The other four were missense mutations (Asp192Val, Ala330Phe, Val364Met, and Arg444Gln), all located in conserved core structures determining proper folding and heme-binding ability of cytochrome P450 molecules. Molecular modeling demonstrated that two of four mutations in positions 330 and 364 were structurally neutral, but Arg444Gln caused significant structural change. Of the four probable mutations, three were missense (Val198Ile, Val320Leu, and Glu499Gly); the other was a base substitution in the noncoding region of exon 1. CONCLUSIONS: The 11 varied CYP1B1 mutations found in 13 unrelated Japanese patients with sporadic occurrence of PCG represent an allelic heterogeneity and may be unique to a specific population.

Effects of protein kinase inhibitor, HA1077, on intraocular pressure and outflow facility in rabbit eyes.

OBJECTIVE: To elucidate the roles of protein kinase in regulating the intraocular pressure (IOP) and outflow facility in rabbit eyes. MATERIALS AND METHODS: A protein kinase inhibitor, 1-(5-isoquinolinesulfonyl)-homopiperazine (HA1077), was used. The IOP and the outflow facility were measured before and after topical, intracameral, or intravitreal administration of HA1077 in rabbits. Western blot analysis was performed to detect the 20-kd light chain of myosin in human trabecular meshwork (TM) cells and bovine ciliary muscle (CM) tissues. The cell morphologic condition and distribution of actin filaments and vinculin in TM cells were studied using cell biology techniques. Carbachol-induced contraction of isolated bovine CM strips following administration of HA1077 was examined in a perfusion chamber. RESULTS: In rabbit eyes, the administration of HA1077 resulted in a significant decrease in IOP in a dose-dependent manner. An increased outflow facility was also observed. Western blot analysis revealed the presence of 20-kd light chain of myosin in human TM cells and bovine CM tissues. In cultured human TM cells, exposure to HA1077 disrupted actin bundles and impaired focal adhesion formation. In addition HA1077 showed relaxation of bovine CM strips. CONCLUSIONS: Use of HA1077 caused a reduction in IOP and an increase in the outflow facility. The results of in vitro experiments suggest that the IOP-lowering effects of HA1077 may be related to the altered cellular behavior of TM cells and relaxation of CM contraction. The results of these studies suggested that protein kinase inhibitors have the potential to be developed into a treatment modality for glaucoma.

Dual effects of interleukin-1beta on N-methyl-D-aspartate-induced retinal neuronal death in rat eyes.

In this study we determine if interleukin-1beta (IL-1beta) modulates N-methyl-D-aspartate (NMDA)-induced retinal damage. Sprague-Dawley rats were anesthetized with inhalation of halothane, after which a single injection of 5 microl of IL-1beta (0.1 to 10 ng/eye) (and/or IL-1 receptor antagonist (IL-1ra)) for experimental eyes was administered. Two days later (or simultaneously), NMDA (20 nmol) was injected into the vitreous space. One week later, each eye was enucleated and transverse sections were subjected to morphometric analysis. Enzyme-linked immunosorbent assay (ELISA) was conducted for the determination of IL-1beta levels in retina. Immunohistochemical and immunoblot studies were also performed. In eyes that received an intravitreal injection of IL-1beta (0.1 to 10 ng/eye), significant thinning of the inner plexiform layer (IPL) was observed (P<0.05). Immunohistochemical and ELISA studies demonstrated upregulated expression of IL-1beta in retinas that had undergone NMDA injection. Treatment with 10 ng of IL-1ra induced a protective effect against NMDA-induced retinal damage. Pretreatment with IL-1beta induced a significant protective effect on NMDA-induced retinal damage. Our studies suggest that IL-1beta induces neuronal cell death directly, as shown by the protective effects of IL-1ra, but has a protective effect on NMDA-induced retinal damage indirectly after an incubation time of at least 2 days.

Inhibitory effects of neurocan and phosphacan on neurite outgrowth from retinal ganglion cells in culture.

PURPOSE: Neurocan and phosphacan are nervous tissue-specific chondroitin sulfate proteoglycans (CSPGs) that are highly expressed in postnatal rat retina. To elucidate potential roles of neurocan and phosphacan on neurite outgrowth from retinal ganglion cells (RGCs), in vitro experiments were conducted with purified RGCs. METHODS: Neurocan and phosphacan were purified from postnatal rat brain by DEAE-column chromatography and subsequent gel chromatography. RGCs were obtained from postnatal rat retinas by a two-step immunopanning procedure using an anti-Thy 1,1 antibody and an anti-macrophage antibody. Neurite outgrowth from RGCs was examined on poly-L-lysine (PLL)-conditioned plates, and PLL-conditioned plates treated with neurocan or phosphacan. RESULTS: Compared with PLL-conditioned plates, neurocan and phosphacan inhibited neurite outgrowth from RGCs at 48 and 72 hours after seeding. When chondroitin sulfate side chains linked to the core proteins were digested by chondroitinase ABC, the inhibitory effect remained, indicating that the core proteins are related to the effect. Furthermore, the digestion of chondroitin sulfate side chains linked to phosphacan core protein significantly promoted the inhibitory effect of phosphacan on neurite outgrowth from RGCs. CONCLUSIONS: Neurocan and phosphacan, which are highly expressed in postnatal rat retina, inhibit neurite outgrowth from postnatal rat RGCs, indicating that these proteoglycans may be inhibitory factors against neurite outgrowth from RGCs during retinal development.

Transforming growth factor-beta 2 levels in aqueous humor of glaucomatous eyes.

PURPOSE: To determine whether clinical characteristics are correlated with increased levels of transforming growth factor-beta 2 (TGF-beta 2) in aqueous humor in glaucomatous eyes. METHODS: Aqueous humor samples were collected from 91 glaucomatous eyes. Included were samples from primary open-angle glaucoma (POAG) in 40 eyes, (pseudo)exfoliation syndrome (EXS) in 18 eyes, primary angle-closure glaucoma (PACG) in 26 eyes and uveitis-related secondary glaucoma (SG) in 7 eyes. TGF-beta 2 in aqueous humor was assessed with a specific-capture ELISA. RESULTS: The mean concentration (+/- standard error) of mature (biologically active) TGF-beta 2 in the aqueous humor of eyes with POAG was 293.6 +/- 33.6 pg/ml, significantly higher than that in eyes with PACG, EXS and SG: 147.5 +/- 28.1, 135.8 +/- 30.2 and 41.0 +/- 10.7 pg/ml, respectively (P = 0.0006, P = 0.0010 and P = 0.0003; analysis of variance). The mean concentration (+/- standard error) of total TGF-beta 2 in the aqueous humor of eyes with POAG was 1647.6 +/- 124.5 pg/ml, not significantly different from that in eyes with PACG, EXS and SG: 1482.9 +/- 148.2, 1442.7 +/- 187.8 and 1929.0 +/- 367.6 pg/ml, respectively. A multivariate analysis using logistic regression showed significant correlations between mature TGF-beta 2 concentration and history of cataract surgery (P = 0.0225) and the use of carbonic anhydrase inhibitors (P = 0.0143). CONCLUSIONS: Our results indicate that increased levels of TGF-beta 2 may play an important role in the pathogenesis of POAG.

Transient intraocular pressure elevation after trabeculotomy and its occurrence with phacoemulsification and intraocular lens implantation.

PURPOSE: To elucidate the characterization of intraocular pressure (IOP) spike after trabeculotomy, and after the combined procedure of phacoemulsification and aspiration (PEA) and intraocular lens (IOL) implantation. METHODS: Included in this study were 39 patients (53 eyes) with primary open-angle glaucoma with IOPs uncontrolled even with anti-glaucoma medication. We conducted a retrospective study for the following two groups: Patients who underwent trabeculotomy alone (25 eyes) and patients undergoing trabeculotomy combined with PEA and implantation of an IOL (28 eyes). RESULTS: In 7 (28%) of the 25 eyes after trabeculotomy alone and 7 (25%) of the 28 eyes after the combined procedure, transient IOP elevation was found postoperatively. The incidence of hyphema-related IOP spike was significantly higher in eyes after trabeculotomy alone (16%) than after the combined procedure (0%). After removal of the blood present in the anterior chamber in eyes with hyphema-related IOP spikes, the IOP levels were well controlled. CONCLUSIONS: Hyphema-related IOP spike is one of the common complications in eyes after trabeculotomy alone, and the combined procedure decreases the incidence of this complication. It is thought that removal of prolonged massive hyphema is effective as treatment for hyphema-related IOP spike.

Effects of rho-associated protein kinase inhibitor Y-27632 on intraocular pressure and outflow facility.

PURPOSE: To elucidate the roles of Rho-associated protein kinase (ROCK) in regulating intraocular pressure (IOP) and outflow facility in the rabbit eye. METHODS: A specific ROCK inhibitor Y-27632 was used. The IOP, the outflow facility, and the pupil diameter were determined before and after the topical, intracameral, or intravitreal administration of Y-27632 in rabbits. Western blot analysis was used to identify specific ROCK isoform in human trabecular meshwork (TM) cells and bovine ciliary muscle (CM) tissues. The cell morphology and distribution of actin filaments and vinculin in TM cells were studied by cell biology techniques. Carbachol (Cch)-induced contraction of isolated bovine CM strips after administration of Y-27632 was measured in a perfusion chamber. RESULTS: In rabbit eyes, administration of Y-27632 resulted in a significant decrease in IOP in a dose-dependent manner. An increase of the outflow facility and pupil size dilation was also observed in Y-27632-treated eyes. Western blot analysis revealed the presence of p160ROCK in human TM cells and bovine CM tissues. In cultured human TM cells, exposure to Y-27632 caused retraction and rounding of cell bodies as well as disruption of actin bundles and impairment of focal adhesion formation. Y-27632 in addition inhibited Cch-induced contraction of isolated bovine CM strips. CONCLUSIONS: Administration of Y-27632 caused a reduction in IOP and an increase in the outflow facility. The in vitro experiments suggest that the IOP-lowering effects of Y-27632 may be related to the altered cellular behavior of TM cells and relaxation of CM contraction. These studies suggest that ROCK inhibitors may have great potential to be developed for treatment of glaucoma and other ocular diseases.

External trabeculotomy for the treatment of steroid-induced glaucoma.

PURPOSE: To investigate the effect of external trabeculotomy on eyes with steroid-induced glaucoma. METHODS: We retrospectively analyzed the surgical results of 14 eyes of seven patients that underwent trabeculotomy for the first surgical procedure. All patients had the history of receiving topical or systemic corticosteroids before the rise of intraocular pressure had been noted. RESULTS: After an average follow-up of 60.6 +/- 33.5 months, in all of the 14 eyes, intraocular pressure was well controlled below or equal to 21 mm Hg at the final examinations. CONCLUSIONS: Surgical results of external trabeculotomy remain effective for a long time. It has been shown that the trabeculotomy can be a useful and effective surgical treatment of patients with steroid-induced glaucoma.

Neuroglycan C, a neural tissue-specific transmembrane chondroitin sulfate proteoglycan, in retinal neural network formation.

PURPOSE: Neuroglycan C (NGC) is a transmembrane chondroitin sulfate proteoglycan present exclusively in central nervous system tissues. In the current study the expression pattern and characterization of NGC during the development of the retina were investigated. METHODS: Expressional changes of NGC mRNAs during rat retinal development were examined by semiquantitative reverse transcription-polymerase chain reaction (RT-PCR). The localization and characterization of NGC core proteins were investigated by immunoblot analysis and immunohistochemistry using an anti-NGC antibody. RESULTS: Immunohistochemical analysis revealed that NGC was highly expressed in the nerve fiber layer (NFL) and inner plexiform layer (IPL) in rat postnatal developing retina. At embryonal stages, NGC immunoreactivities were faint. In contrast, at postnatal developmental stages (approximately postnatal day [P]7), intense immunoreactivity was observed in the NFL and IPL, where active dendrite branching was observed, and conventional synapses began to be formed. As retinal layer differentiation proceeded (from P14 to P42), immunoreactivities in the inner retinal layers gradually became fainter. Immunoblot and semiquantitative RT-PCR analyses showed that the peak level of NGC expression occurred on approximately P7 and P14. Glycosylation of the NGC core protein changed as the retinal layers matured. In immunoelectron microscopic analysis, NGC immunoreactivity was located on the axonal membranes of neuronal cells in the postnatal retina, whereas immunoreactivity was reduced on membranes at the adult stage. In retinal ganglion cells in vitro, NGC was highly localized in their spiny budding neurites. CONCLUSIONS: The results show spatiotemporal expression patterns of NGC, and suggest that it plays a role in the formation of neural networks in retinal development.

Neuroprotective effects of brain-derived neurotrophic factor in eyes with NMDA-induced neuronal death.

PURPOSE: To determine if brain-derived neurotrophic factor (BDNF) has a neuroprotective effect against N-methyl-D-aspartate (NMDA)-induced cell death in retina. METHODS: NMDA was injected into the vitreous of rat eyes. NMDA-induced neuronal death was measured by morphometric analyses on cell counts of ganglion cell layer cells and thickness of retinal layers. Also, we conducted additional experiment using retrograde labeling with a fluorescent tracer (Fluoro-Gold) for exact counting of retinal ganglion cells (RGCs). In addition, intravitreal glutamate levels were measured with the use of a high-performance liquid chromatography (HPLC) system. RESULTS: Morphometric analysis of retinal damage in NMDA-injected eyes showed that BDNF could protect inner retinal cells from glutamate receptor-mediated neuronal death. Also, counts of RGCs labeled with a fluorescent tracer showed that BDNF could protect RGCs from glutamate receptor-mediated neuronal death. Furthermore, measurements of intravitreal glutamate levels indicated an increase in this excitatory amino acid in the vitreous after NMDA injection. CONCLUSIONS: Exogenous BDNF can protect inner retinal cells (possible RGCs and amacrine cells) from NMDA-induced neuronal death. However, increased intravitreal glutamate levels in response to NMDA-mediated neurotoxicity may augment retinal degeneration.

Secondary glaucoma associated with crystalline lens subluxation.

PURPOSE: To elucidate the clinical characteristics of secondary glaucoma associated with subluxation of the crystalline lens. SETTING: Department of Ophthalmology and Visual Sciences, Kyoto University Graduate School of Medicine, Kyoto, and Department of Ophthalmology, Tenri Hospital, Nara, Japan. METHODS: This retrospective study comprised 14 eyes of 13 patients with uncontrolled intraocular pressure (IOP) and lens subluxation. The subluxated lens was extracted through surgery. RESULTS: Angle closure caused by the subluxated lens was complicated in 3 eyes. In the remaining 11 eyes, uncontrolled IOP elevation was found despite the presence of deep anterior chambers and wide open angles. A mean of 14.1 months +/- 13.7 (SD) after cataract surgery, IOP was well controlled (lower than 21 mm Hg) in all 14 eyes. Mean IOP was 15.4 +/- 2.2 mm Hg at the final examination. Complications included transient vitreous hemorrhage in 5 eyes, choroidal detachment in 2 eyes, and retinal tears in 1 eye. CONCLUSION: Lens extraction surgery was effective in controlling IOP in eyes with secondary glaucoma associated with lens subluxation.

Novel mutations in the myocilin gene in Japanese glaucoma patients.

Myocilin is a gene responsible for juvenile onset primary open angle glaucoma (POAG) mapped as the GLC1A locus and, many mutations have been reported worldwide. Some mutations were found not only in patients with juvenile onset POAG, but also in patients with late onset POAG and in patients with normal tension glaucoma. To investigate the mutation prevalence in Japan, we performed a mutation analysis in 140 unrelated Japanese patients. We have identified the 10 sequence variants, of which four were highly probable for disease-causing mutations (Arg46ter, Arg158Gln, Ile360Asn, and Ala363Thr), and six polymorphisms (Gln19His, Arg76Lys, Asp208Glu, Val439Val, Arg470His, and Ala488Ala). Thus, myocilin mutations were found at the rate of 4/140 (2.9%) probands, similar to previous reports with other ethnic populations.

Upregulated expression of neurocan, a nervous tissue specific proteoglycan, in transient retinal ischemia.

PURPOSE: Neurocan, a nervous tissue-specific chondroitin sulfate proteoglycan synthesized primarily by neurons, is expressed abundantly in developing rat retina, whereas it is rarely expressed in adult rat retinas. This study investigated the reexpression of neurocan in a pathologic condition of adult rat retina. METHODS: Transient retinal ischemia was produced by occlusion of the retinal artery for 60 minutes. After transient retinal ischemia, neurocan expression was investigated by reverse transcription-initiated polymerase chain reaction (RT-PCR), immunohistochemistry, and immunoblot analysis. RESULTS: Semiquantitative analysis using RT-PCR revealed that mRNA expression for neurocan increased at 24 hours after reperfusion. Furthermore, on immunoblot analysis using an anti-neurocan antibody, MAb 1G2, the intensity of the 220-kDa band as well as the 150-kDa band increased markedly at 24 and 72 hours after reperfusion. The 220-kDa band was predominant at 24 hours after reperfusion, whereas the intensity of the 150-kDa band became almost the same as that of the 220-kDa band at 72 hours after reperfusion. Immunohistochemical analysis revealed that upregulated neurocan immunoreactivity was associated with glial Müller cells. CONCLUSIONS: Thus, upregulated expression of neurocan in transient retinal ischemia was demonstrated. Furthermore, the immunohistochemical analysis revealed that the upregulated expression of neurocan is derived from Müller cells, although it has been thought that neurocan is synthesized by neurons so far. The neurocan expression by Müller cells suggests that this proteoglycan plays a role in the damage and repair processes in diseased retina.