Reconstruction of a nonfunctional trabeculectomy bleb using an amniotic membrane-wrapped silicone sponge to treat refractory glaucoma.

BACKGROUND: This study was conducted to verify the usefulness of nonfunctional trabeculectomy bleb reconstruction using a silicone sponge wrapped with amniotic membrane. Its purpose was to allow aqueous humor to flow from the flap to the posterior orbital space. METHODS: Seven consecutive patients who had undergone two or more surgeries in one eye for refractory glaucoma followed by our operation were included in this study. Conjunctival adhesion to the sclera was detached with a limbus-based conjunctival incision, followed by reopening the former trabeculectomy flap. A 1.5 × 12 mm silicone sponge used for retinal detachment surgery was wrapped three to four times with amniotic membrane, placed longitudinally on the sclera, and fixed with 10-0 nylon sutures. The anterior end of the amniotic membrane was fixed underneath the scleral flap with sutures, and the conjunctival wound was closed. We periodically checked the intraocular pressure (IOP) and for complications. Follow-up periods ranged from 15 to 30 months (average 19.4 months). Surgical success was defined as a final IOP of ≤ 21 mmHg with or without additional treatment. We defined failure as an IOP of > 21 mmHg on the second of two consecutive visits after the first 4 weeks, or the need for additional glaucoma surgery. RESULTS: Surgery was successful in five of the seven eyes, although bleb needling was performed in two eyes and amniotic membrane patch covering for early aqueous leakage was needed in one eye. In four of the five successful eyes, IOP was well controlled for longer than the period between the previous and present surgeries. One of the unsuccessful eyes, with neovascular glaucoma, had high IOP with hyphema followed by phthisis of the eyeball. The other, with aqueous leakage via the conjunctival wound, required trabeculectomy in a different area. There were no other complications. CONCLUSIONS: Reconstruction of the nonfunctional trabeculectomy bleb using a silicone sponge wrapped with amniotic membrane can be a useful strategy for treating refractory glaucoma.

Genetic variants of FZD4 and LRP5 genes in patients with advanced retinopathy of prematurity.

PURPOSE: Retinopathy of prematurity (ROP) is a complex disease with a genetic predisposition, but little is known about its genetic background. It has a clinical resemblance to familial exudative vitreoretinopathy (FEVR), a hereditary disease characterized by defects in the development of retinal vessels. Several studies have suggested that mutations in the causative genes for FEVR may account for a proportion of advanced ROP, but conflicting data have also been reported for some variants. To address the possibility of genetic involvement of FEVR genes in ROP, we performed comprehensive sequence analyses of 53 Japanese patients with advanced ROP for the FEVR-causing genes. METHODS: Peripheral blood DNA was obtained from 53 patients referred to our hospitals for retinal surgery. Polymerase chain reaction followed by direct sequencing of the coding regions of the known FEVR-causing genes (FZD4, LRP5, TSPAN12, and NDP) and a noncoding exon of the NDP gene was performed. Possible pathogenicity of the sequence changes were analyzed by orthologous protein sequence alignment and by computational predictions. RESULTS: We identified six different nonsynonymous DNA variants in the coding region of either the FZD4 gene (p.H69Y, p.R127H, and p.Y211H) or the LRP5 gene (p.R1219H, p.H1383P, and p.T1540M) in seven patients. The corresponding codons of these changes were highly conserved among species, and these changes were predicted to be pathogenic by at least two of four computational prediction programs. No such changes were found in the TSPAN12 and NDP genes. CONCLUSIONS: Six possibly pathogenic variants of FZD4 or LRP5 were found in seven advanced ROP patients. Although these variants do not yet provide definitive evidence that they are causal, the results imply a role of the FZD4 and LRP5 genes in the development of advanced ROP.

IOP measured by dynamic contour tonometry correlates with IOP measured by Goldmann applanation tonometry and non-contact tonometry in Japanese individuals.

PURPOSE: The purpose of this study was threefold. We sought to compare the intraocular pressure (IOP) measured by dynamic contour tonometry (DCT) with that measured by Goldmann applanation tonometry (GAT) and noncontact tonometry (NCT). We also examined the influence of central corneal thickness (CCT) and corneal curvature radius (CCR) on the IOP measurements. Last, we investigated the factors that could affect the ocular pulse amplitude (OPA) measurements. METHODS: Seventy-four patients with no history of intraocular surgery were enrolled in this study. We measured IOP by DCT, GAT, and NCT, and the CCT, CCR, and axial length (AL) in the right eye of each patient. We also measured OPA by DCT. We subsequently analyzed the correlation of IOP measurements between GAT and DCT and between NCT and DCT. We also examined the influence of CCT, CCR, and AL on IOP readings by the 3 tonometers. In addition, we investigated the factors that could affect the OPA measurements. RESULTS: The mean IOP measured by DCT was 2.8 mm Hg higher than that by GAT and 3.2 mm Hg higher than that by NCT. This difference was greater with thinner CCT in the lower IOP group than in the higher IOP group. IOP measurements by both GAT and NCT significantly correlated with CCT; however, IOP measurement by DCT did not correlate with CCT. No significant correlations were shown between the IOP measured by each of the 3 tonometers and either CCR or AL. OPA measurements positively correlated with age, IOP measurement by DCT, and pulse pressure. CONCLUSIONS: IOP measured by DCT correlates with IOP measured by GAT or NCT with a roughly 3.0 mm Hg higher value, and these differences were greater in the patients with a thinner CCT. IOP measurements by both GAT and NCT significantly correlated with CCT; however, IOP measurement by DCT did not correlate with CCT. Our findings also indicate that OPA measured using DCT shows a positive correlation with patient age, IOP measurement by DCT, and pulse pressure.

Transcriptional regulation of activating transcription factor 4 under oxidative stress in retinal pigment epithelial ARPE-19/HPV-16 cells.

PURPOSE: Oxidative stress plays an important role in the pathogenesis of various ocular diseases such as retinopathy, glaucoma, and age-related macular degeneration. Activating transcription factor 4 (ATF4) is induced by various stressors, including endoplasmic reticulum (ER) and oxidative stress, and ATF4 expression is regulated translationally through the PERK pathway of eIF2α phosphorylation. Transcriptional regulation of the ATF4 gene under oxidative stress was investigated in human papillomavirus 16 (HPV-16)-transformed retinal pigment epithelial ARPE-19/HPV-16 cells. METHODS: Retinal pigment epithelial cells, trabecular meshwork cells, and corneal endothelial cells were treated with anoxia and thapsigargin (TG). Gene expression of ATF4 and nuclear factor (erythroid-derived 2)-like 2 (Nrf2) and transcription factors was investigated by Western blot analysis, reporter assays, chromatin immunoprecipitation (ChIP) assays, and small interfering (si)RNA strategies. Cellular sensitivity to oxidative stress was determined. RESULTS: The expression of two transcriptional factors, ATF4 and Nrf2, was significantly induced by anoxia and TG. The Nrf2 regulator Keap1 was downregulated by anoxia. Downregulation of Nrf2 abolished ATF4 expression. On the other hand, downregulation of Keap1 enhanced the expression of both Nrf2 and ATF4. The promoter activity of ATF4 was transactivated by the co-transfection of Nrf2 expression plasmids and reduced by the transfection of Nrf2-specific siRNA. The ChIP assays demonstrated that Nrf2 bound to the promoter of the ATF4 gene. Nrf2 downregulation nearly abolished the ATF4 induction by anoxia and TG. Consistent with these findings, the promoter activity of ATF4 was augmented by treatment with TG, HCA, H(2)O(2), and anoxia. However, stress induction of ATF4 promoter activity was observed, even when a mutation was introduced into the antioxidant-responsive elements site. Furthermore, stress induction of the ATF4 promoter was completely abolished when the 5' untranslated region of the ATF4 gene was deleted. Downregulation of ATF4 rendered ARPE-19/HPV-16 cells sensitive to oxidative stress. CONCLUSIONS: These results suggest that the stress induction of ATF4 is significantly regulated transcriptionally through a Nrf2-dependent mechanism and may be a double-edged sword in the pathogenesis of various retinopathies.

Angiographic changes in iris and iridocorneal angle neovascularization after intravitreal bevacizumab injection.

OBJECTIVE: To evaluate the effects of the intravitreal (IV) injection of bevacizumab on anterior segment neovascularization using anterior segment angiography. METHODS: We observed 1 eye with iris and iridocorneal angle neovascularization and 3 with neovascular glaucoma from 4 patients with diabetic retinopathy in 3 eyes and central retinal vein occlusion in 1 eye. Two healthy eyes from 2 other patients served as control eyes. Three eyes, including 1 normal eye, were examined by iris angiography; the other eyes underwent iridocorneal angle angiography with fluorescein (FA) and indocyanine green (IA) using a Heidelberg Retina Angiograph 2. After angiography, 4 eyes with neovascularization were treated with IV bevacizumab (1.25 mg per 0.05 mL) and underwent angiography once more 4 to 6 days after treatment. RESULTS: Iris angiography with indocyanine green revealed many iris vessels, but not dye leaking, in both normal and glaucomatous eyes, and the angiography with fluorescein showed intensive vessel leakage in the iris as well as iridocorneal angle neovascularization, but not in normal eyes. Angle angiography revealed vessel structures with indocyanine green and intensive leakage with fluorescein in the iris and showed iridocorneal angle neovascularization and neovascular glaucoma, whereas no vessel structures appeared with IA or FA in the normal eye. After IV bevacizumab injection in eyes with neovascularization, the vascular structure did not change with IA, but dye leakage remarkably decreased with FA in the iris and angle. However, newly formed vessels in the iris and iridocorneal angle seemed to disappear on slitlamp examination. CONCLUSION: Intravitreal injection of bevacizumab effectively reduces vascular permeability, whereas newly formed vessels are still present in the iris and iridocorneal angle.

Amniotic Membrane Transplantation for Repair of Leaking Glaucoma Filtering Blebs with Scleral Perforation.

Leaking glaucoma filtering blebs with scleral perforation were successfully repaired in two patients using amniotic membrane transplantation. The amniotic membrane was placed into the subconjunctival space to cover the perforated scleral area. The edge of the limbal conjunctiva was sutured to the peripheral cornea with conjunctival advancement over the amniotic membrane. The bleb leaks were successfully closed. In addition, good and functioning filtration was maintained during a follow-up period of 12 months in both cases. Amniotic membrane transplantation may be effective for the surgical management of high risk of leaking glaucoma blebs with scleral perforation.

Histology of fibrovascular membranes of proliferative diabetic retinopathy after intravitreal injection of bevacizumab.

PURPOSE: The purpose was to study the histology of the fibrovascular membranes in proliferative diabetic retinopathy (PDR) with an intravitreal injection of bevacizumab. METHODS: Light and electron microscopic studies were performed on surgical specimens obtained during a pars plana vitrectomy from 6 PDR eyes after intravitreal injection of bevacizumab. The patients had preoperatively received no or scant retinal photocoagulations. The presence and distribution of CD34 was assessed as a marker of vascular endothelium using immunostaining. The presence of vascular endothelial growth factor was stained with a method of immunostaining. As controls, we examined 7 surgical specimens from 7 PDR eyes obtained during pars plana vitrectomy without bevacizumab therapy. All control patients had preoperatively received full or nearly full pan retinal photocoagulations. RESULTS: Light microscopy showed that the CD34-positive vascular endothelial cells formed capillarylike structures in the fibrovascular membranes of all 13 PDR eyes. Vascular endothelial growth factor was positively stained in the vascular endothelium of both groups; however, the number of vascular endothelial growth factor-positive vascular endothelial cells significantly decreased in the fibrovascular membranes with intravitreal injection of bevacizumab. Electron microscopy showed the newly formed vascular endothelial cells with junctional complex in both groups. CONCLUSION: The vascular endothelial cells with decreased expression of vascular endothelial growth factor are still present in the fibrovascular membranes of patients with PDR after intravitreal injection of bevacizumab.

Nipradilol and timolol induce Foxo3a and peroxiredoxin 2 expression and protect trabecular meshwork cells from oxidative stress.

PURPOSE: Oxidative stress plays an important role in pathogenesis of glaucoma. The purpose of this study is to investigate the novel effect of antiglaucoma drugs on the expression of antioxidant peroxiredoxins of trabecular meshwork (TM) cells. METHODS: The expression of the peroxiredoxin family was investigated using immortalized TM cell lines. Cells were treated with antiglaucoma drugs and analyzed for the expression of peroxiredoxin, and cellular sensitivity to oxidative stress. Furthermore, the effect of antiglaucoma drugs on the molecular regulation of the expression of peroxiredoxin was examined using a reporter assay and siRNA strategy. RESULTS: Glaucomatous TM cells highly express peroxiredoxin 2 when compared with normal TM cells. Nipradilol and timolol, but not latanoprost, induce the expression of peroxiredoxin 2 through the activation of the Foxo3a transcription factor. TM cells showed reduced sensitivity to H(2)O(2) when cells were treated with either nipradilol or timolol, but not with latanoprost. In addition, both Foxo3a and PRDX2 expression were enhanced by drug-induced signal transduction through its receptor. CONCLUSIONS: These results indicate that both nipradilol and timolol possess a novel mechanism of action and function as potent protective agents against oxidative stress.

Tip60 is regulated by circadian transcription factor clock and is involved in cisplatin resistance.

Histone modification is important for maintaining chromatin structure and function. Recently, histone acetylation has been shown to have a critical regulatory role in both transcription and DNA repair. We report here that expression of histone acetyltransferase (HAT) genes is associated with cisplatin resistance. We found that Tip60 is overexpressed in cisplatin-resistant cells. The expression of two other HAT genes, HAT1 and MYST1, did not differ between drug-sensitive and -resistant cells. Knockdown of Tip60 expression rendered cells sensitive to cisplatin but not to oxaliplatin, vincristine, and etoposide. Tip60 expression is significantly correlated with cisplatin sensitivity in human lung cancer cell lines. Interestingly, the promoter region of the Tip60 gene contains several E boxes, and its expression was regulated by the E-box binding circadian transcription factor Clock but not by other E-box binding transcription factors such as c-Myc, Twist, and USF1. Hyperacetylation of H3K14 and H4K16 was found in cisplatin-resistant cells. The microarray study reveals that several genes for DNA repair are down-regulated by the knockdown of Tip60 expression. Our data show that HAT gene expression is required for cisplatin resistance and suggest that Clock and Tip60 regulate not only transcription, but also DNA repair, through periodic histone acetylation.

[A case of secondary retinal detachment caused by ocular toxocariasis].

PURPOSE: We report a case with bullous retinal detachment secondary to ocular toxocariasis. CASE: A 68-year-old man, who was a professional dog breeder, visited an ophthalmologist because of visual field defect in the left eye, and was referred to our clinic. The patient had bilateral cataract and bullous retinal detachment in the left eye. Fundus examinations after cataract surgery revealed no break but a white mass in the temporal lower peripheral retina of the left eye. Initial treatment with systemic corticosteroids was ineffective. Retinal detachment was treated by retinal cryocoagulation, scleral buckling, and subretinal fluid drainage. Subretinal fluid obtained during the operation showed high antibody titer for Toxocara canis. CONCLUSION: Ocular toxocariasis can cause bullous retinal detachment. To confirm the diagnosis, examination of the antibody titer of Toxocara canis in the subretinal fluid is useful.

Ultrastructure of the trabecular meshwork in secondary glaucoma eyes after intravitreal triamcinolone acetonide.

PURPOSE: To study the histology of the trabecular meshwork of eyes with glaucoma by intravitreal injection of triamcinolone acetonide (TA). DESIGN: Two cases report. PARTICIPANTS/METHODS: A 68-year-old Japanese man with branch retinal vein occlusion and a 48-year-old Japanese woman with uveitis were treated by cataract surgery, intraocular lens implantation, and TA-assisted pars plana vitrectomy. At the end of surgery, TA suspension (4 mg) was intravitreously injected. During the follow-up period, the intraocular pressure (IOP) of the patients increased over 30 mm Hg even with full medication. Trabeculectomy was performed at 4 months after TA injection in case 1 and at 6 months in case 2, and intraocular pressure returned to the normal range in both cases. Light and electron microscopic studies of the resected trabecular tissue were carried out. RESULTS: The histology showed minimal deposition of extracellular matrix in the trabecular meshwork in case 1. Case 2 showed the beginnings of deposition of extracellular matrix including fingerprintlike material in the trabecular meshwork with decreased intertrabecular spaces. CONCLUSIONS: The ultrastructural changes in the trabecular meshwork of eyes with glaucoma after treatment with intravitreal TA might resemble those with glaucoma after topical corticosteroid treatment.

[Effects of implementing clinical pathways for the care of patients undergoing ophthalmic surgery for cataract, glaucoma, and vitreoretinal disorder].

PURPOSE: To evaluate the actual use of clinical pathways and variances, and compare the length of hospital stay for surgery of cataract, glaucoma, and vitreoretinal disorder. METHODS: We designed eight types of clinical pathways for the treatment of cataract, glaucoma, and retinal-vitreous disease. We performed 102 phacoemulsifications and intraocular lens (IOL) implantations, 19 glaucoma or combined trabeculotomy and phacoemulsification/IOL, and 69 retinal-vitreous surgeries during a 1-year period from February 2002. We compared the length of the hospital stay before and after clinical pathway implementation. RESULTS: We applied the clinical pathways to 102 eyes (100%) of 67 patients undergoing phacoemulsification/IOL, to 17 eyes (89.5%) of those undergoing glaucoma surgery, and to 69 eyes (100%) of those undergoing retinal-vitreous surgery. The vaiances occurred in 20 eyes (29.9%) of 67 phacoemulsification/IOLs, 6 eyes (31.6%) of glaucoma, and 24 eyes (34.2%) of retinal-vitreous surgery. The length of hospital stay was shortened in phacomulsification/IOL after clinical pathway implemenation: 7.8 +/- 3.3 to 6.7 +/- 2.5 (mean +/- standard deviation) days. Glaucoma patients had a significantly shorter stay, from 16.4 +/- 5.0 to 12.6 +/- 3.3 days Mann-Whitney U test ; p = 0.032), and the hospital tay for retinal-vitreous surgery was shortened rom 22.8 +/- 11.1 to 17.9 +/- 6.2 days (p = 0.001). CONCLUSIONS: The application of clinical pathways resulted in substantially reduced hospital stay.

GM-CSF activates RhoA, integrin and MMP expression in human monocytic cells.

Monocyte migration is one of the key events occurring in the early stage of atherosclerosis. This process includes monocytic adhesion to and penetration through the arterial intima. In such an environment, many factors stimulate the monocytes to enhance integrin activation and extracellular matrix degradation. To investigate the coordinative operation of these two events in relation to monocyte migration, we paid particular attention to the effects of granulocyte-macrophage colony-stimulating factor (GM-CSF) on monocytes in terms of RhoA activation and matrix metalloproteinase (MMP) expression. RhoA and integrin clustering were activated by GM-CSF, monocyte chemoattractant protein-1 (MCP-1) and platelet-derived growth factor-BB (PDGF-BB) in human monocytic cell lines. Furthermore, enhancement of migration was observed with stimulation by MCP-1 and PDGF-BB. Granulocyte-macrophage colony-stimulating factor did not enhance the migration, even though it activated RhoA and integrin. However, GM-CSF is known to stimulate monocytes to express MCP-1, suggesting the presence of an indirect mechanism for GM-CSF-mediated migratory activity. In contrast, only GM-CSF enhanced the expression of MMP-1 and MMP-9. These results provide evidence that GM-CSF has multiple functions enhancing monocytic migration via RhoA and integrin activation, and via MMP expression.

The rarity of Charles Bonnet syndrome.

Charles Bonnet syndrome (CBS) is characterized by complex visual hallucinations in otherwise psychologically normal people. Estimates of the prevalence of CBS in different samples vary from a small percentage (around 1%), to a relatively large percentage (about 10%). The purpose of the present study is to determine whether CBS is rare or not. One-thousand ophthalmologic and optometric outpatients at a university hospital were consecutively screened by a questionnaire to identify patients possibly experiencing visual hallucinations. The mean corrected visual acuity in the best eye was 1.1. Those who positively responded to the questionnaire were further investigated to determine whether their symptoms were consistent with CBS. As a result, the prevalence of CBS was 0.5% (5/1000). In subclass analyses, the prevalence was 3 of 372 (0.8%) in the low vision group, 2 of 346 (0.6%) in the elderly, and 1 of 120 (0.8%) in both conditions. These were not significantly different from each other or from the overall prevalence (0.5%). This low prevalence of CBS in our subjects may be due to their relatively good visual acuity because previous studies with high prevalence of CBS investigated patients with a visual acuity of less than 0.3. The prevalence of CBS may be low in patients with these particular characteristics, and this syndrome seems to be rare in even ophthalmologic and optometric patients if they do not have seriously low vision. Further studies are needed to investigate the prevalence of CBS in general population.

Cornea with Peters' anomaly: perturbed differentiation of corneal cells and abnormal extracellular matrix in the corneal stroma.

PURPOSE: We examined histopathologically the anterior ocular segment including the cornea and lens of an eye which had been enucleated in a patient with Peters' anomaly because of untreatable corneal perforation. Special effort was made to differentiate the corneal stromal and endothelial cells, and the stromal extracellular matrix. METHODS: Light microscopy, with hematoxylin and eosin staining, and transmission electron microscopy were employed. RESULTS: Corneal endothelial cells and Descemet's membrane were not detected in the central cornea, where there were immature cells with a fibroblastic configuration. The inner surface of the peripheral cornea was covered with cells containing pigment granules in the cytoplasm. Cell density in the central corneal stroma was relatively high. The diameter of the stromal collagen fibrils was not uniform. A mature collagen fibril-free area was also seen in the central corneal stroma. CONCLUSIONS: Differentiation of neural crest-derived cells in corneal stroma and endothelium might have been perturbed in the cornea of this patient with Peters' anomaly, inducing the defect in the corneal endothelium and the qualitative and quantitative abnormalities of the extracellular matrix.

Neovascularization in the anterior segment of the rabbit eye by experimental anterior ischemia.

PURPOSE: To investigate the effects of anterior ischemia accompanied by neither retinal nor choroidal ischemia on the anterior segment of the eye. METHODS: Both long posterior ciliary arteries in the right eye of 14 rabbits were directly cauterized with an electric coagulator. The eyes were enucleated 1, 2, 4, 7, 9 or 14 days after cauterization, then fixed with 4% paraformaldehyde. Semi-thin sections were studied by light microscopy. Several sections were stained with Griffonia simplicifolia lectin, which bound specifically to mammalian vascular endothelium. Other specimens were examined immunohistochemically for vascular endothelial growth factor (VEGF) protein. The tissue specimens of the first postoperative day were studied for expression of VEGF mRNA by in situ hybridization. RESULTS: Atrophy of the iris and ciliary body was seen after the second postoperative day. Corneal neovascularization appeared after 7 days. Neovascularization on the anterior surface of the iris and in the trabecular meshwork was detected after the ninth postoperative day. The proliferative tissues with newly formed vessels obstructed the iridocorneal angle 14 days after the treatment. There was no histological change in either the retina or choroid. Immunohistochemically, VEGF protein was detected in the epithelial and vascular cells of the iris on the first and fourth postoperative day. Expression of VEGF mRNA was detected in the epithelial cells of the ciliary body on the day following the treatment. CONCLUSIONS: Anterior segment ischemia, when unaccompanied by retinal ischemia, causes neovascularization in the cornea, iris and trabecular tissue.