Abstract:

Abstract: PURPOSE: To evaluate the association between the third high-reflectance band on high-resolution optical coherence tomography (OCT), fundus autofluorescence (AF), and kinetic perimetry results in patients with typical retinitis pigmentosa (RP). DESIGN: Retrospective, observational case series. METHODS: Thirty-four patients with typical RP who were referred to our institute were examined, with a diagnosis made by full-field electroretinography. We evaluated the fundus AF and the third high-reflectance band by high-resolution OCT, both qualitatively and quantitatively. We investigated whether the vertical length of the AF diameter or the third high-reflectance band correlated with Goldmann kinetic perimetry results. RESULTS: We classified three types of abnormal fundus AF: ring AF, central AF, and the absence of both patterns. In eyes with ring AF, the length of the third high-reflectance band was almost equal to the diameter of the abnormal ring AF with significant correlation (P < .001), whereas the band length did not correlate with the diameter of the visual field (P = .237). Eyes with central AF did not have a continuous third high-reflectance band. In eyes with neither ring nor central AF, the length of the third high-reflectance band correlated with the AF length and the diameter of the visual field (P = .024 and P < .001, respectively). CONCLUSIONS: A novel classification based on the fundus AF and the third high-reflectance band determined by OCT suggests different patterns of pathogenesis in the retinal pigment epithelium and photoreceptor degeneration in the progression of RP.

Abstract: BACKGROUND AND PURPOSE: More than half of the retinitis pigmentosa (RP) cases are genetically simplex or multiplex. To date, 37 causative genes of RP have been identified; however, the elucidation of gene defects in simplex or multiplex RP patients/families remains problematic. The aim of our study was to identify the genetic causes of RP in patients with unknown or non-Mendelian inheritance. METHODS AND RESULTS: Since 2003, 52 simplex RP patients, 151 patients from 141 multiplex RP families, and six sporadic patients with retinal degeneration were studied. A total of 108 exons of 30 RP-causing genes that harboured the reported mutations were screened by an efficient denaturing high performance liquid chromatography (dHPLC) based assay. Aberrant fragments were subsequently analysed by automatic sequencing. Twenty-six mutations, including two frameshift mutations, one single amino acid deletion, and 23 missense mutations, were identified in 28 probands (14.07%). Eighteen mutations have not been reported to date. Three pairs of combined mutations in different genes were identified in two sporadic cases and one multiplex family, indicating the possibility of novel digenic patterns. Of the 23 missense mutations, 21 were predicted as deleterious mutations by computational methods using PolyPhen, SIFT, PANTHER, and PMut programs. CONCLUSION: We elucidated the mutation spectrum in Japanese RP patients and demonstrated the validity of the mutation detection system using dHPLC sequencing for genetic diagnosis in RP patients independent of familial incidence, which may provide a model strategy for identifying genetic causes in other diseases linked to a wide range of genes.

Abstract: BACKGROUND/AIMS: To assess the outcomes of 23-gauge sutureless transconjunctival vitrectomies (TSV), as compared with 25-gauge TSV in macular hole surgeries. METHODS: A retrospective, consecutive, interventional case series of 47 eyes with idiopathic macular holes treated by 23- or 25-gauge TSV were analysed. RESULTS: The operative time was 37.2 (SD 8.9) min with 23-gauge TSV and 34.2 (8.7) min with 25-gauge TSV (p = 0.388). The anatomical success rate was 96% with 23-gauge TSV and 92% with 25-gauge TSV (p>0.999). The logarithm of the minimum angle of resolution of best-corrected visual acuity (BCVA) at the sixth postoperative month was 0.19 (0.16) with 23-gauge TSV and 0.19 (0.25) with 25-gauge TSV (p = 0.521). Postoperative improvement in BCVA was comparable between the two TSVs. IOP on postoperative day 1 was lower with 25-gauge TSV (12.3 (4.9) mm Hg) than with 23-gauge TSV (17.4 (5.8) mm Hg) (p = 0.036). Complications included retinal break, intraoperative bleeding and slippage of the infusion cannula with 23-gauge TSV, while retinal detachment and postoperative hypotony occurred in the 25-gauge TSV group (p = 0.570). CONCLUSION: 23-gauge TSV appears to be as safe and effective as 25-gauge TSV in macular hole surgery.

Abstract: We have approached the role of cellular stress in neurodegenerative diseases caused by polyglutamine expansion (polyQ) in the context of Spinocerebellar ataxia type 7 (SCA7) that includes retinal degeneration. Using the R7E mouse, in which polyQ-ataxin-7 is specifically over-expressed in rod photoreceptors, we previously showed that rod dysfunction correlated to moderate and prolonged activation of the JNK/c-Jun stress pathway. SCA7 retinopathy was also associated with reduced expression of rod-specific genes, including the transcription factor Nrl, which is essential for rod differentiation and function. Here, we report that R7E retinopathy is improved upon breeding with the JunAA knock-in mice, in which JNK-mediated activation of c-Jun is compromised. Expression of Nrl and its downstream targets, which are involved in phototranduction, are partially restored in the JunAA-R7E mice. We further show that c-Jun can directly repress the transcription of Nrl. Our studies suggest that polyQ-induced cellular stress leads to repression of genes necessary for neuronal fate and function.

Abstract: PURPOSE: To diagnose an atypical retinal degenerative disease with choroidal neovascularization by means of gene diagnosis. CASE: A 47-year-old woman had good visual acuity at the first examination. She had scattered chorioretinal degeneration and pigmentation in the peripheral retina. There was a symmetrical visual field defect in the upper and temporal periphery in both eyes. Seven years later, choroidal neovascular membrane (CNV) developed in the fovea of her left eye and visual acuity deteriorated to 0.4 in this eye. Optical coherence tomography revealed type 2 CNV with minimal subretinal fluid. Fluorescein angiography showed very little leakage from the CNV. We used denaturing high performance liquid chromatography(DHPLC) to perform gene diagnosis and found a peripherin/RDS gene mutation of Gly167-Ser. CONCLUSION: Our case had moderate peripheral retinal degeneration with CNV. It is possible that cases like this tend to be misdiagnosed as AMD (age-related macular degeneration) or CNV with high myopia. Evaluation of the gene mutation was useful for diagnosis in this case.

Abstract: PURPOSE: To report novel mutations in the GRK1 gene in Japanese patients with Oguchi disease. DESIGN: Observational case report. METHODS: Two unrelated Japanese patients with Oguchi disease were examined. After informed consent was obtained, the coding regions of SAG and GRK1 were analyzed by direct sequencing. RESULTS: Although no mutation was found in SAG, two novel homozygous mutations in GRK1, c.1079 del T and c.1408-1412 CCCCC to CCC, were identified. Both mutations are expected to generate null alleles of GRK1. CONCLUSIONS: The authors found two different novel mutations in Japanese patients. The results indicate that a considerable number of GRK1 mutations exist in the Japanese population.

Abstract: Although recent studies revealed chondroitinase ABC (ChABC), an enzyme that degrades chondroitin sulfate proteoglycans, promotes CNS regeneration in vivo, the usefulness of its application for transplantation is not clear. We investigated if treatment with ChABC can promote synapse formation between graft and host neurons following retinal transplantation. Dissociated retinal cells were prepared from neonatal Nrl-GFP transgenic mice in which rod photoreceptors and their progenitor cells are labeled with GFP. Each cell suspension with or without ChABC (Nrl/ChABC group and Nrl group, respectively) was injected subretinally into the eyes of mice following chemically induced photoreceptor degeneration. The survival and functional integration of the transplanted photoreceptors were examined by histologically and electrophysiologically. Up to 4 weeks after transplantation, almost all the grafted GFP+ photoreceptor cells were widely distributed at the outer margin of the host retina where the photoreceptor layer was located originally. In the Nrl/ChABC group, 33.6% of the GFP+ photoreceptors elaborated neurites horizontally or vertically, and 4.6% elaborated neurites toward the retina. These neurites extended over the glial seal at the graft-host interface, and established synaptic contacts with neurons in the host retina as determined by confocal microscopy and three-dimensional analysis. Although 30.7% cells (p = 0.68) elaborated neurites in the Nrl group, only 1.2% cells (p < 0.05) projected neurites towards the host tissue and synaptic contacts were rare. Our results illustrate the potential utility of ChABC for enhancing synaptogenesis between transplanted neurons and host retina.

Abstract: The Maf-family transcription factor Nrl is a key regulator of photoreceptor differentiation in mammals. Ablation of the Nrl gene in mice leads to functional cones at the expense of rods. We show that a 2.5-kb Nrl promoter segment directs the expression of enhanced GFP specifically to rod photoreceptors and the pineal gland of transgenic mice. GFP is detected shortly after terminal cell division, corresponding to the timing of rod genesis revealed by birthdating studies. In Nrl-/- retinas, the GFP+ photoreceptors express S-opsin, consistent with the transformation of rod precursors into cones. We report the gene profiles of freshly isolated flow-sorted GFP+ photoreceptors from wild-type and Nrl-/- retinas at five distinct developmental stages. Our results provide a framework for establishing gene regulatory networks that lead to mature functional photoreceptors from postmitotic precursors. Differentially expressed rod and cone genes are excellent candidates for retinopathies.

Abstract: Photoreceptor loss causes irreversible blindness in many retinal diseases. Repair of such damage by cell transplantation is one of the most feasible types of central nervous system repair; photoreceptor degeneration initially leaves the inner retinal circuitry intact and new photoreceptors need only make single, short synaptic connections to contribute to the retinotopic map. So far, brain- and retina-derived stem cells transplanted into adult retina have shown little evidence of being able to integrate into the outer nuclear layer and differentiate into new photoreceptors. Furthermore, there has been no demonstration that transplanted cells form functional synaptic connections with other neurons in the recipient retina or restore visual function. This might be because the mature mammalian retina lacks the ability to accept and incorporate stem cells or to promote photoreceptor differentiation. We hypothesized that committed progenitor or precursor cells at later ontogenetic stages might have a higher probability of success upon transplantation. Here we show that donor cells can integrate into the adult or degenerating retina if they are taken from the developing retina at a time coincident with the peak of rod genesis. These transplanted cells integrate, differentiate into rod photoreceptors, form synaptic connections and improve visual function. Furthermore, we use genetically tagged post-mitotic rod precursors expressing the transcription factor Nrl (ref. 6) (neural retina leucine zipper) to show that successfully integrated rod photoreceptors are derived only from immature post-mitotic rod precursors and not from proliferating progenitor or stem cells. These findings define the ontogenetic stage of donor cells for successful rod photoreceptor transplantation.

Abstract: NRL (neural retina leucine zipper) is a key basic motif-leucine zipper (bZIP) transcription factor, which orchestrates rod photoreceptor differentiation by activating the expression of rod-specific genes. The deletion of Nrl in mice results in functional cones that are derived from rod precursors. However, signaling pathways modulating the expression or activity of NRL have not been elucidated. Here, we show that retinoic acid (RA), a diffusible factor implicated in rod development, activates the expression of NRL in serum-deprived Y79 human retinoblastoma cells and in primary cultures of rat and porcine photoreceptors. The effect of RA is mimicked by TTNPB, a RA receptor agonist, and requires new protein synthesis. DNaseI footprinting and electrophoretic mobility shift assays (EMSA) using bovine retinal nuclear extract demonstrate that RA response elements (RAREs) identified within the Nrl promoter bind to RA receptors. Furthermore, in transiently transfected Y79 and HEK293 cells the activity of Nrl-promoter driving a luciferase reporter gene is induced by RA, and this activation is mediated by RAREs. Our data suggest that signaling by RA via RA receptors regulates the expression of NRL, providing a framework for delineating early steps in photoreceptor cell fate determination.

Abstract: The success of functional retinal cell transplantation has been limited by the low efficiency of the transplanted cell integration into the host retina. Given that the extracellular matrix (ECM) is thought to inhibit entry and axonal outgrowth of grafted neural cells into the host retina, modulation of the ECMs in the host environment may overcome this limitation. Here, we demonstrate that matrix metalloprotease-2 (MMP-2) expression is associated with the high migratory potential of adult rat hippocampus-derived neural stem cells compared with retinal progenitor cells. In addition, MMP-2, as well as its reported inducers concanavalin A and 17beta-estradiol, can trigger the migration of retinal progenitor cells into explanted retinas. Inhibitors of MMP-2 suppressed these effects. Intense cell migration is not required for photoreceptor transplantation; however, the environment that allows the transplanted cells to integrate is most important. Migration of the transplanted cells is a good index of the acceptance of grafted cell of the host tissue. Strategies modulating the environment by MMP-2 stimulation may provide an advance in the development of retinal transplantation.

Abstract: Mutations in the retinitis pigmentosa GTPase regulator (RPGR) gene account for almost 20% of patients with retinitis pigmentosa. Most mutations are detected in alternatively spliced RPGR-ORF15 isoform(s), which are primarily but not exclusively expressed in the retina. We show that, in addition to the axoneme, the RPGR-ORF15 protein is localized to the basal bodies of photoreceptor connecting cilium and to the tip and axoneme of sperm flagella. Mass spectrometric analysis of proteins that were immunoprecipitated from the retinal axoneme-enriched fraction using an anti-ORF15 antibody identified two chromosome-associated proteins, structural maintenance of chromosomes (SMC) 1 and SMC3. Using pulldown assays, we demonstrate that the interaction of RPGR with SMC1 and SMC3 is mediated, at least in part, by the RCC1-like domain of RPGR. This interaction was not observed with phosphorylation-deficient mutants of SMC1. Both SMC1 and SMC3 localized to the cilia of retinal photoreceptors and Madin-Darby canine kidney cells, suggesting a broader physiological relevance of this interaction. Additional immunoprecipitation studies revealed the association of RPGR-ORF15 isoform(s) with the intraflagellar transport polypeptide IFT88 as well as microtubule motor proteins, including KIF3A, p150Glued, and p50-dynamitin. Inhibition of dynein function by overexpressing p50 abrogated the localization of RPGR-ORF15 to basal bodies. Taken together, these results provide novel evidence for the possible involvement of RPGR-ORF15 in microtubule organization and regulation of transport in primary cilia.

Abstract: Regenerative medicine constitutes a potentially promising therapy for blind people suffering from retinal degenerative diseases such as retinitis pigmentosa and age-related macular degeneration. For the realization of retinal regeneration, it is necessary to establish 1) a method to produce functional photoreceptor cells in vitro and 2) successful transplantation of the donor cells to connect their axons to the recipient secondary neurons so that they can function properly. The results of experimental transplantation of human retinal photoreceptor cells from cadaveric eyes or of fetal retinal cells into the retina of RP patients have not been satisfactory, but encouraging enough to indicate that the transplantation of developing retinal cells may have beneficial results. Recently, attempts have been made to generate photoreceptor-like cells from stem cells, but it remains to be seen whether they are in fact photoreceptor cells. It is therefore important to fully understand the mechanisms involved in the development of these cells, and to characterize them not only by transcriptome but also by functional analysis.

Abstract: A number of PCR-based in situ hybridization (ISH) techniques have been reported to facilitate the procedure. However, those techniques require additional gene specific primers with RNA polymerase binding site. We developed a new PCR-based ISH technique without extra gene-specific primers. We amplified gene specific PCR products with regular gene-specific primer pairs. Special linker, including T7 RNA polymerase binding site, was adapted to amplified PCR products. Secondary PCR was performed with T7 primer, and forward or reverse primer, used for the first PCR to prepare template DNA for RNA transcription. We were able to generate sense and anti-sense probes for ISH in a day. Recently, real-time PCR and ISH are required to validate microarray results quantitatively and qualitatively. This technique can be expected to facilitate the high-throughput validation of transcripts detected by microarrays.

Abstract: PURPOSE: To establish lines of transgenic mice that express Cre-recombinase in M- or S-cone photoreceptors for generating cone photoreceptor-specific (conditional) mutants. METHODS: Five kilobases of 5' upstream sequence of the mouse red-green (M) opsin gene or 0.5 kb of the mouse blue (S) opsin gene was cloned into a Cre-expression plasmid. Transgenic mice were generated and characterized, and appropriate lines were established. The Cre-transgenic mice were crossed with ROSA26-lacZ mice (containing floxed beta-galactosidase gene) and analyzed to determine Cre-recombinase activity. RESULTS: Immunofluorescence study showed successful targeting of Cre-recombinase expression to cone photoreceptors. Double staining with anti-Cre antibody and anti-M- or anti-S-opsin antibody revealed specificity of Cre expression in M-opsin- and/or S-opsin-positive photoreceptors. Mating with ROSA26-lacZ mice demonstrated that Cre-recombinase was functionally active in M- or S-cones. CONCLUSIONS: Lines of transgenic mice that specifically express functional Cre-recombinase in M- or S-cones were established in this study. Because mutations in several widely expressed genes lead to photoreceptor degeneration, these transgenic mice should be valuable in generating conditional mutants to investigate the function of various genes specifically in cone photoreceptors.

Abstract: Many mammalian retinas are rod-dominant, and hence our knowledge of cone photoreceptor biology is relatively limited. To gain insights into the molecular differences between rods and cones, we compared the gene expression profile of the rod-dominated retina of wild type mouse with that of the cone-only retina of Nrl(-/-) (Neural retina leucine zipper knockout) mouse. Our analysis, using custom microarrays of eye-expressed genes, provided equivalent data using either direct or reference-based experimental designs, confirmed differential expression of rod- and cone-specific genes in the Nrl(-/-) retina and identified novel genes that could serve as candidates for retinopathies or for functional studies. In addition, we detected altered expression of several genes that encode cell signaling or structural proteins. Prompted by these findings, additional real-time PCR analysis revealed that genes belonging to the Bmp/Smad and Wnt/Ca(2+) signaling pathways are expressed in the mature wild type retina and that their expression is significantly altered in the Nrl(-/-) retina. Chromatin immunoprecipitation analysis of adult retina identified Bmp4 and Smad4, which are down-regulated in the Nrl(-/-) retina, as possible direct transcriptional targets of Nrl. Consistent with these studies, Bmp4 and Smad4 are expressed in the mature rod photoreceptors of mouse retina. Modulation of Bmp4 and/or Smad4 by Nrl may provide a mechanism for integrating diverse cell signaling networks in rods. We hypothesize that Bmp/Smad and Wnt/Ca(2+) pathways participate in cell-cell communication in the mature retina, and expression changes observed in the Nrl(-/-) retina reflect their biased utilization in rod versus cone homeostasis.

Abstract: PURPOSE. To systematically explore changes in gene expression in the retina of monkeys with laser-induced glaucoma and to validate the microarray data on eyes with experimental glaucoma. METHODS. Glaucoma was induced in the right eye of four monkeys by repeated argon laser photocoagulation of the trabecular meshwork. The left eye served as the control. Retinas were isolated from glaucomatous and control eyes 30 days after photocoagulation. Gene expression changes were analyzed by human microarray chips which displayed a total of 9182 elements including Expression Sequence Tag (EST) clones. Changes in the expression of some genes were further confirmed by real-time PCR analysis. Immunohistochemical studies to examine protein expression of some gene products were also done for several genes that showed up- or downregulation by the microarray analysis. RESULTS. Two eyes with mild glaucoma and two with severe glaucoma were produced. In the mild and severe glaucomatous retina, the number of upregulated genes was 45 and 18, and the number of downregulated genes was 17 and 21, respectively. The number of genes that were up- or downregulated was 0.7% of all the genes examined. The real-time PCR analysis confirmed expression changes of some genes found in the microarray analysis. Ceruloplasmin was one of the upregulated genes, and it was found by immunohistochemical analyses to be expressed in Müller cells. CONCLUSIONS. Gene expression profiles in laser-induced glaucomatous monkey retinas were determined, and only a very small population of genes was up- or downregulated in glaucomatous eyes. Upregulation of ceruloplasmin protein was found in the Müller cells.

Abstract: OBJECTIVE: Excitatory amino acid (EAA) toxicity seems to be an important mechanism of neuronal cell death after cerebral infarction. We examined the inhibitory effects of neuronal cell death caused by EAA in vitro by means of adenoviral gene transfer of neurotrophic basic fibroblast growth factor (bFGF) and antiapoptotic Bcl-xL. METHODS: Recombinant adenoviral vectors expressing human bFGF gene with secretory signals of interleukin-2 and human Bcl-xL gene were constructed. Primarily cultured rat neuronal cells were treated with glutamate to cause EAA, and the neuroprotective effects of gene transfer by these adenoviral vectors were investigated at several time points of infection. RESULTS: Each adenoviral infection to primarily cultured neuronal cells exhibited neuroprotective effects against EAA caused by glutamate. Both gene transfer of bFGF with secretory signal and Bcl-xL transfer to neuronal cells exhibited the synergistic neuroprotective effects against EAA. These effects were most prominent with gene transfer 4 hours before glutamate insult; gene transfer performed simultaneously with and up to 4 hours after the insult exhibited definite neuroprotective effects. CONCLUSION: These experiments revealed marked neuroprotective effects of adenoviral gene transfer of bFGF and Bcl-xL into neuronal cells in vitro. The findings may lead to new approaches for treating occlusive cerebrovascular disease.

Abstract: AIMS: To evaluate a new delivery system of 5-fluorouracil (5-FU) using 5-fluorocytosine (5-FC) as a prodrug and cytosine deaminase induced in vitro and in vivo. METHODS: Fibroblastic cells from rabbit Tenon's capsule were cultured. The cells were exposed to 5-FU and 5-FC with or without cytosine deaminase induced by recombinant adenovirus. In the in vitro study, cell proliferation and DNA synthesis were assessed by MTS, BrdU assay. The effect of 5-FC removal after the treatment of 5-FC and cytosine deaminase induction was also assayed. In the in vivo study cells with or without cytosine deaminase induction were transplanted into the subconjunctival space of mice, followed by eye drops of 1000 microg/ml of 5-FC three times a day. The mice were sacrificed at days 1, 5, and 10, then the cells transplanted were evaluated. RESULTS: Cell proliferation was inhibited by exposure to 5-FU in a dose dependent manner; however, up to 1000 microg/ml of 5-FC did not affect cell proliferation. Cell proliferation was inhibited by exposure to 5-FC in a time dependent manner with induction of cytosine deaminase following infection of recombinant adenovirus. When 5-FC was removed 3 or 6 days after the treatment, the cells grew again. The effect was reproduced in the in vivo model of subconjunctival cellular proliferation although 5-FC was administrated as eye drops. There were no cases with corneal erosion. CONCLUSION: Cell proliferation was inhibited by co-exposure of 5-FC and cytosine deaminase. This new delivery system may merit controlled delivery of 5-FU after filtering surgery.

Abstract: A female with von Hippel-Lindau (VHL) disease type 2A first presented with erythrocytosis at the age of 9 years. This patient revealed multiple paragangliomas at age 22. After the removal of tumors, a retinal hemangioblastoma developed. Our diagnosis of VHL disease type 2A was confirmed. Moreover, systemic examination showed a duodenal somatostatinoma. Frequent and long-term monitoring is important for patients with pheochromocytomas or paragangliomas, and a screening for VHL disease and other hereditary cancer syndromes is recommended. Recognition of neuroendocrine tumors as a manifestation of VHL disease permits earlier diagnosis and improves prognosis.

Abstract: BACKGROUND: Heterozygous mutations of the bestrophin gene are associated with Best macular dystrophy (BMD). The bestrophin gene is specifically expressed in the retinal pigment epithelium. BMD is a hereditary form of macular degeneration that may develop subretinal neovascularisation similar to the wet type of age-related macular degeneration (AMD). PURPOSE: To study whether mutations of the bestrophin gene occur in non-familial Japanese AMD patients. METHODS: A total of 85 non-familial AMD patients (average age 67.5 years; 71 male, 14 female) diagnosed by indocyanine green angiography were screened. Among them, 69 patients (81 %) were classified as having wet type AMD. Genomic DNA was purified from the total blood and used as the template for polymerase chain reaction (PCR). All the exons of bestrophin gene were amplified by PCR. Mutation analysis was performed by SSCP using the ABI Prism 310 Genetic Analyzer (Perkin Elmer). Nucleotide sequence was determined by direct sequencing of the PCR amplicons. As the control, 105 non-AMD patients (average age 62.0 years; 52 male, 53 female) were screened by the same method. RESULTS: Only one AMD patient had a specific polymorphism in exon 2, but no mutations leading to amino acid substitutions were found. In exon 2 and 3, two further polymorphisms were detected in all AMD patients as well as normal controls. CONCLUSION: No mutations were found in the bestrophin gene in nonfamilial Japanese patients with AMD or in normal controls.

Abstract: PURPOSE: To compare the retinal nerve fiber layer thickness in eyes with idiopathic macular holes and age-matched normal controls using scanning laser polarimeter. METHODS: The retinal nerve fiber layer thickness was measured in 40 eyes of 40 consecutive patients with idiopathic macular hole (stage 1, 10 eyes; stage 2, eight eyes; stage 3, 15 eyes; stage 4, seven eyes) and 40 eyes of 40 age-matched normal controls with a scanning laser polarimeter. The retinal nerve fiber layer thickness within a 10-pixel-wide ellipse located concentrically with the disk and located 1.5-disk diameters from the center of the disk was measured. The mean overall retinal nerve fiber layer thickness of the peripapillary retina, four 90-degree quadrants, and 16 equal sectors of every 22.5 degrees was calculated for both groups. The retinal nerve fiber layer thickness in the two groups was statistically compared. RESULTS: The mean retinal nerve fiber layer thickness measurement for the overall peripapillary retina and for three of the four 90-degree quadrants was not significantly different between the two groups. However, the temporal 90-degree quadrant was significantly thinner in the macular hole group (47.2 versus 54.6 microm, P =.026). For the 16 sectors of 22.5 degrees, the lower three sectors of the four sectors in the temporal quadrant were thinner in the macular hole group (P <.05). CONCLUSIONS: The retinal nerve fiber layer thickness of the papillomacular area is thinner in eyes with idiopathic macular hole than that in normal eyes. The progressive thinning of the retinal nerve fiber layer thickness as the stage of the macular hole advances may suggest that surgery should be done at the earliest stage.

Abstract: Adenovirus-mediated ex vivo gene transfer of basic fibroblast growth factor (bFGF), a new strategy for the treatment of chronic vascular occlusive disease, was examined in a rabbit model of hind limb ischemia. The left femoral artery was completely excised to induce an ischemic state in the hind limb of male rabbits. Simultaneously, a skin section was resected from the wound, and host fibroblasts were cultured. The cultured fibroblasts were infected with adenovirus vector containing modified human bFGF cDNA with the secretory signal sequence (AxCAMAssbFGF) or LacZ cDNA (AxCALacZ). At 21 days after femoral artery excision, the gene-transduced fibroblasts were administered through the left internal iliac artery. The fibroblasts significantly accumulated in the ischemic hind limb, and the AxCAMAssbFGF-treated cells secreted bFGF for less than 14 days without elevation of systemic bFGF level. At 28 days after cell administration, calf blood pressure ratio, angiographic score, capillary density of muscle tissue and blood flow of the left internal iliac artery were determined, and animals with AxCAMAssbFGF-treated cells showed significantly greater development of collateral vessels, as compared with those with AxCALacZ-treated cells. These findings suggest that adenovirus-mediated ex vivo gene transfer of bFGF was effective for improvement of chronic limb ischemia.

Abstract: We constructed two replication-deficient recombinant adenovirus vectors coding human basic fibroblast growth factor (bFGF), one with and one without the interleukin-2 (IL-2) secretory signal sequence and examined their neurotrophic effects on primary neuronal cells in vitro. The primary neuronal cells were successfully infected at a high efficiency with the adenovirus vectors. bFGF protein was detected in the culture medium of the neurons infected with both these vectors. The cells infected with the bFGF-expressing adenovirus containing the IL-2 signal sequence showed 2- to 10-fold higher levels of secretion levels than cells infected with the native bFGF-expressing adenovirus alone. Both bFGF-expressing vectors augmented the survival of primary neuronal cells in an in vitro culture, compared with a mock infection or control virus infection. Notably, the cells infected with the bFGF-expressing adenovirus containing the IL-2 signal sequence were markedly enhanced cell survival in the early phase of the culture, compared with the control cells and even those infected with the bFGF-expressing adenovirus without the IL-2 signal sequence. However, in the late phase of neuronal culture, neither viral vector could support the cell survival. In contrast the co-infection of the bFGF-expressing vector with a Bcl-xL-expressing vector was extremely effective on neuronal survival.

Abstract: PURPOSE: To investigate a possible role of the nitric oxide (NO)-cGMP signal transduction system in phagocytosis of rod outer segments (ROS) by cultured retinal pigment epithelial (RPE) cells. METHODS: Primary cultures of RPE cells from 10-day-old Brown Norway rats were used to study the phagocytosis of ROS by these cells. Phagocytosis of ROS was evaluated with or without an inhibitor of nitric oxide synthase (NOS), N(G)-nitro-L-arginine (L-NNA), and the reverse effects of L-NNA by L-arginine and 8-bromo-cGMP on phagocytosis were also studied. NO-associated cGMP production by RPE cells was monitored during phagocytosis using L-NNA. NOS activity was assayed in RPE cells and ROS to locate the source of NO. RESULTS: Phagocytosis of ROS was inhibited by L-NNA but not by D-NNA. L-NNA inhibited the ingestion in a dose-dependent manner, but not the binding of ROS. The inhibition was reversed by L-arginine and also by an NO donor, SIN-1. RPE cells challenged with ROS showed increased cGMP activity, which was significantly reduced by L-NNA and again restored by an overdose of L-arginine. NOS activity was found in RPE cells but not in ROS. CONCLUSIONS: Our data show that cGMP plays a role in the ingestion phase of ROS phagocytosis by RPE cells via a cGMP second-messenger system.

Abstract: Cerebral ischemic disease often causes morbidity and mortality, while the induction of new blood vessels is expected to provide a therapeutic effect in this occlusive cerebrovascular disease. In this study, we utilized two replication-deficient adenoviral vectors containing cDNA from basic fibroblast growth factor (bFGF), a well-known angiogenic factor, and examined whether biological angiogenic activity of adenovirally gene-transferred bFGF could be observed in the rat brain. One vector contained native cDNA from bFGF without the secretory signal sequence and the other contained the same cDNA fused with an interleukin-2 secretory signal sequence. After ventricular administration of these viral vectors, gene-transferred cells demonstrated a high immunoreactivity against the anti-bFGF antibody and a remarkably high concentration of bFGF was detected in the cerebrospinal fluid. A semiquantitative analysis of angiogenic activity revealed that bFGF gene transfer induced angiogenesis in normal rat brains, with a more pronounced angiogenic effect seen with the vector of a secreted form than with the vector without a secretory signal sequence. These results suggest that bFGF gene transfer using these adenoviral vectors might be useful for the treatment of ischemic cerebrovascular disease.

Abstract: PURPOSE: To report that optical coherence tomography as early as 24 hours after macular hole surgery shows anatomic configuration of the closed macular holes. METHOD: In a prospective study, seven eyes of seven consecutive patients with stage 3 or 4 idiopathic macular hole underwent surgery. Optical coherence tomography was performed preoperatively and at 24, 48, and 72 hours after the surgery. RESULTS: Optical coherence tomography images could be obtained on four out of the seven eyes at 24 hours after surgery. These images showed anatomic configuration of the closed macular holes. Surgical success was confirmed in all of the eyes when the gas was completely absorbed. CONCLUSION: Optical coherence tomography revealed anatomic configuration of surgically closed macular holes within 24 hours after successful surgery.

Abstract: Bcl-xL is a Bcl-2-related gene that regulates programmed cell death in a bcl-2-independent fashion. It is expressed in tissues containing long-surviving postmitotic cells, such as neurons in adult brains. To investigate the possibility of gene therapy for transferring this anti-apoptotic gene into the neuron for the treatment of vascular occlusive or neurodegenerative diseases, we examined the effect of a replication-deficient recombinant adenovirus vector coding human Bcl-xL gene on the augmentation of the survival of primarily-cultured rat neuronal cells in vitro. Immunoblot analysis revealed that primarily-cultured neuronal cells were successfully infected and transferred with this gene by recombinant adenovirus vector with high transduction efficiency. Bcl-xL gene transfer to the primarily-cultured neurons could prevent these cells from cell death.

Abstract: PURPOSE: To evaluate the abilities of recombinant adenovirus carrying the basic fibroblast growth factor (bFGF) gene to (1) produce bFGF protein in vitro and (2) rescue retinal photoreceptors in Royal College of Surgeons (RCS) rats in vivo. METHODS: Cultured human retinal pigment epithelial cells were infected with one of the following two replication-deficient adenoviral vectors that drive inserted genes by beta-actin promoter with cytomegalovirus enhancer: AxCAJSbFGF, which expresses the human bFGF gene, and AxCAlacZ, carrying the cDNA of bacterial beta-galactosidase as a viral control. These viruses and recombinant bFGF protein were also injected into the subretinal space of RCS rats at the age of 21 days. The production of bFGF was evaluated by an immunohistochemical method in vitro and in vivo. The secretion of bFGF produced in vitro was quantified by an enzyme-linked immunosorbent assay. The thickness of the outer nuclear layer (ONL) as a marker of photoreceptor cell rescue was estimated at 2, 28, and 56 days after the injections. RESULTS: AxCAJSbFGF produced human bFGF protein effectively both in vitro and in vivo. The semiquantitative analysis of ONL thickness revealed a significant protective effect of AxCAJSbFGF and the recombinant bFGF protein injection up to 56 days after injection. CONCLUSIONS: These results demonstrate that a recombinant adenoviral vector can achieve the transfer of bFGF gene in vitro and have a protective effect for photoreceptor cells in vivo. Gene therapy with a bFGF-expressing recombinant adenoviral vector may provide a new strategy with which to target retinal degenerative diseases.

Abstract: PURPOSE: To test the ability of a mutant herpes simplex virus (HSV) hrR3 to inhibit growth of Y79 human retinoblastoma in vitro and in vivo. METHODS: Cultured Y79 cells were infected with multiplicities of infection (MOI) ranging from 0.004 to 0.1 of hrR3. Surviving cells were counted using trypan blue dye exclusion. Using X-gal staining, expression of the lacZ gene was examined in vitro on day 3 postinfection to evaluate viral replication. Nude mice harboring Y79 tumors subcutaneously received an intraneoplasmic injection of 5 x 10(7) plaque-forming units of hrR3. The tumor sizes were measured weekly. Expression of the lacZ gene was also examined on one week postinfection. RESULTS: There are 31% and 13% cells surviving in cultured Y79 cells infected by hrR3 at an MOI of 0.1 on days 3 and 5 postinfection respectively compared to those of mock-infected cells. Also more than 70% of Y79 cells were stained with X-gal at an MOI of 0.1 which demonstrated active viral replication in vitro. Virus-treated subcutaneous tumors were smaller than control tumors (p<<0.05, Student's t-test) on days 14, 21, and 28 postinfection. Positive X-gal staining was also observed in the tumor nodule which was challenged with this viral vector. CONCLUSIONS: We have demonstrated that hrR3 is capable of inhibiting Y79 tumor growth both in cell culture and in nude mice. These data suggest that gene therapy using this mutant HSV vector can be a new supplementary therapeutic modality for retinoblastoma.

Abstract: The transcription factor E2F regulates the expression of several genes concerned with cell growth. The ability to inhibit transcription by blocking E2F expression has great potential in the treatment of proliferative disorders. The effect of double-stranded phosphorothioate oligonucleotides containing E2F transcription factor cis element, a so called 'decoy' has examined on the growth of cultured human Tenon's fibroblastic cells. Human Tenon's fibroblastic cells were cultured and challenged by E2F decoy coated with the Hemagglutinating virus of Japan (HVJ) cationic liposomes (HVJ-CL). The outcome was evaluated using fluorescence microscopy, RT-PCR and growth assays. HVJ-CL facilitated the transfer of external oligonucleotides to cultured human Tenon's fibroblastic cells. The E2F decoy, transferred by HVJ-CL, inhibited simultaneously the expression of the mRNAs of several cell cycle related genes such as c-myc, cdc2, proliferative cell nuclear antigen, and dehydrofolate reductase. Entry into S phase was also reduced to 42.7% of the positive control by the E2F decoy. The total increase of DNA at four days was reduced to 59.7% of the positive control by 5 microM and 29.9% by 15 microM of E2F decoy. It is concluded that gene therapy using the E2F transcription factor offers a potential therapeutic modality for the treatment of proliferative disorders such as proliferative vitreoretinopathy and fibrosis following filtering surgery.

Abstract: The optic nerve head in severely myopic eyes may be particularly vulnerable to glaucomatous damage. To study this hypothesis, we examined 122 primary open-angle glaucoma eyes with fair to good control of the intraocular pressure and a sign of baseline optic nerve damage. Then, parameters for the progression of the visual field defects were evaluated by multivariate analysis. A high mean intraocular pressure (p = 0.007) and a large refractive error (p = 0.023) were significant risk factors for subsequent visual field loss. A high baseline cup-to-disk ratio (p = 0.100) was a marginal risk factor. Nonsignificant parameters included patient age (p = 0.692), the use of beta-adrenergic antagonists (p = 0.384), gender (p = 0.831) and left versus right side (p = 0.977). When the refractive error was used to subclassify patients into severely myopic (< or = -4 dpt), mildly myopic (-0.25 to -4 dpt), or emmetropic and hyperopic (> or = 0 dpt), only severe myopia was a significant risk factor for progressive visual field loss. Severe myopia, but not mild myopia, is a significant risk factor for subsequent visual field loss in patients with primary open-angle glaucoma.

Abstract: Trabeculotomy ab externo has been demonstrated to be effective in controlling intraocular pressure (IOP) in adult patients with either primary open-angle glaucoma or pseudoexfoliation syndrome. We evaluated the surgical outcome of 60 eyes with either primary open-angle glaucoma or pseudoexfoliation syndrome that underwent combined trabeculotomy ab externo and cataract extraction. All patients were at least 40 years old, and were followed for at least 1 year. At the final examination, IOP was well controlled (21 mm Hg or less) in 54 (90%) of the 60 eyes, with or without medication. Also, "overall success" (ie, stabilization of IOP, visual field, and optic nerve status) was achieved in 49 (81.7%). Complications included fibrin exudation (22%), transient IOP elevation (17%), early perforation of the probe into the anterior chamber (10%), and detachment of Descemet's membrane (5%). We recommend combined trabeculotomy ab externo and cataract extraction in selected cases of glaucoma with coexisting cataract. For cases in which the target IOP level is in the low teens, or for patients who may not tolerate postoperative fluctuations in IOP, we do not recommend trabeculotomy ab externo. Also, in eyes that have normal-tension glaucoma, or that have already sustained severe damage to the optic nerve, visual dysfunction caused by glaucomatous changes may progress even after successful combined trabeculotomy ab externo and cataract extraction.

Abstract: BACKGROUND: We previously reported the effectiveness of goniosynechialysis and trabeculotomy ab externo for adult-onset glaucoma. In this study, we performed non-filtering surgery on patients with primary angle-closure glaucoma and studied the long-term outcome of this treatment. METHODS: Included in this study were 35 eyes of 25 patients with primary angle-closure glaucoma, each of which had an intraocular pressure greater than 20 mmHg with maximal tolerated antiglaucoma medication, even after laser iridotomy or surgical iridectomy. Of these 35 eyes, 22 underwent trabeculotomy and 13 underwent goniosynechialysis. All patients were followed up for at least 18 months. RESULTS: In 21 (95%) of 22 eyes after trabeculotomy, and in 12 (92%) of 13 eyes after goniosynechialysis, intraocular pressures were well controlled at or below 21 mmHg at the final examination. However, in two of the 21 eyes in which trabeculotomy was a success, and in four of the 12 eyes in which goniosynechialysis was successful, the procedure had to be repeated before adequate control of pressure was achieved. CONCLUSION: Our results show that intraocular pressure in most cases of primary angle-closure glaucoma can be controlled by restructuring of the physiologic aqueous outflow route by means of goniosynechialysis or trabeculotomy, and that filtering surgery is not necessary.

Abstract: OBJECTIVE: To elucidate long-term surgical outcome of trabeculotomy ab externo in the treatment of developmental glaucoma. PATIENTS: Included in this retrospective study are 116 eyes of 71 patients with developmental glaucoma. We classified patients into three groups based on their age: congenital (33 eyes), existing before age 2 months; infantile (31 eyes), occurring from ages 2 months to 2 years; and juvenile (52 eyes), age 2 years or older. RESULTS: A life-table analysis showed that the total success probabilities at 5 and 10 years with one or more trabeculotomy ab externo operations were, respectively, 92.5% +/- 2.7% and 76.5% +/- 6.2%. The success probability of patients with congenital glaucoma (60.3% +/- 15.9%) was significantly lower than it was for those with infantile (96.3% +/- 3.6%) or juvenile (76.4% +/- 7.5%) glaucoma (P < .01 for both). CONCLUSIONS: Surgical results of trabeculotomy ab externo remain effective for a long time. Congenital glaucoma has the worst prognosis, and infantile glaucoma has a better prognosis than does juvenile glaucoma.

Abstract: OBJECTIVES: Retrospective and prospective studies examined the surgical effects in lowering intraocular pressure levels of trabeculotomy ab externo in adult eyes with either primary open angle glaucoma or pseudoexfoliation syndrome. We report the results of primary trabeculotomy as an intervention for glaucoma. PATIENTS: Included in the retrospective study were 357 eyes of 227 patients with primary open angle glaucoma and 82 eyes of 65 patients with pseudoexfoliation syndrome, each of which underwent trabeculotomy ab externo alone and none of which had a history of laser or surgical treatment for ocular disease. Included in the prospective study were 33 eyes of 22 patients with primary open angle glaucoma and 17 eyes of 15 patients (older than 40 years) with pseudoexfoliation syndrome. RESULTS: A life-table analysis for the retrospective study showed that the final success probability (mean +/- SE) at 5 years after surgery was 73.5% +/- 6.3% in eyes with pseudoexfoliation syndrome, which is significantly better than 58.0% +/- 3.1% in eyes with primary open angle glaucoma (P < .05). Also, the higher the preoperative intraocular pressure levels are, the less likely that the postoperative intraocular pressure levels will be brought under control (P < .01). Surgical outcome in the prospective study demonstrated success in 27 (79%) of 34 eyes after 3 years and in 16 (64%) of 25 eyes after 5 years, with medication. Complications included Descemet's membrane detachment (one eye), cyclodialysis (one eye), and decreased visual acuity due to progression of cataract (four eyes). CONCLUSION: The surgical results of trabeculotomy ab externo remain effective in controlling intraocular pressure levels for a long time in selected patients. It thus appears that trabeculotomy ab externo can be considered as an alternative choice of surgical treatment in some cases of glaucoma.