Potential participation of CTRP6, a complement regulator, in the pathology of age related macular degeneration.

PURPOSE: To investigate the localized expression of C1q/tumor necrosis factor related protein (CTRP) 6 in human age-related macular degeneration (AMD) retinal tissues. EXPERIMENTAL STUDY DESIGN: 4 AMD and 3 non-AMD whole eyes of Caucasian donors were used. Eyecups were excised at Eye Bank CorneaGen, Inc. METHODS: To elucidate the effects of CTRP6, C3b was measured by an enzyme-linked immunosorbent-like assay. CFB versus CTRP6 competitive binding assay was applied to clarify the inhibition by CTRP6 of C3bBb complex formation. The cornea, iris, lens, and vitreous were removed and the eyes were cut into a posterior eye-cup including the retina, choroid, and sclera. Six-µm-thick serial sections of frozen samples underwent hematoxylin-eosin (HE) staining and indirect immunohistochemical staining using primary antibodies, anti-CTRP6, -CTRP5, -CTRP10, -Complement factor H (CFH) and -Clusterin (CLU). Results The two in vitro studies confirmed that CTRP6 has an inhibitory effect on alternative pathways of complement (APC) function and that the molecular target of CTRP6 is the inhibition of the formation of C3bBb. Localized expression for CTRP6 and CFH was found in the drusen of the AMD eyes, both associated with APC inhibition, CLU associated with membrane-attack complex (MAC) inhibition, and CTRP5 associated with retinal degeneration. CONCLUSION: The localized expression of CTRP6 in the drusen of AMD eyes may open a new insight into the possible involvement of APC regulatory factors in the pathogenesis of AMD, together with the known CFH so far analyzed solely as an APC inhibitor.

Ex vivo Comparison of Intraocular Pressure Fluctuation during Pars Plana Vitrectomy Performed Using 25- and 27-Gauge Systems.

INTRODUCTION: The purpose of this study was to compare intraoperative intraocular pressure fluctuation using different aspiration systems and 25- and 27-gauge vitreous surgery probes. METHODS: Ex vivo, pars plana, 25- and 27-gauge vitreous surgery was performed on 4 porcine eyes, and IOP fluctuations were evaluated. We performed 3-port vitrectomy using the Constellation® Vision or the EVA® Phaco-Vitrectomy system. Each 20-s experiment was conducted 5 times for each set of conditions, each with the same substituted balanced salt solution. Real-time intraoperative intraocular pressure measurement was performed at the distal end of the infusion tube. Intraocular pressure was measured during core vitrectomy, core vitrectomy with fluid aspiration, peripheral vitreous shaving with scleral indentation, and fluid-gas exchange. The Mann-Whitney U test was used to evaluate statistical significance. RESULTS: Mean ± standard deviation intraoperative intraocular pressure fluctuation during 25- and 27-gauge core vitrectomy was 15.9 ± 1.6 and 11.9 ± 1.4 mm Hg, respectively (p < 0.05), using the Constellation system; 23.2 ± 1.4 and 14.1 ± 0.7 mm Hg, respectively (p < 0.001), using the EVA vacuum mode; and 15.0 ± 0.5 and 11.5 ± 1.4 mm Hg, respectively (p < 0.05), using the EVA flow mode. The smallest intraoperative intraocular pressure fluctuations during core vitrectomy with fluid aspiration, peripheral vitreous shaving with scleral indentation, and fluid-gas exchange were all achieved using the 27-gauge EVA flow mode; these values were 14.2 ± 0.4, 35.7 ± 0.9, and 6.4 ± 0.2 mm Hg, respectively. CONCLUSION: Regardless of the aspiration system, intraoperative intraocular pressure fluctuation was lower during 27-gauge than during 25-gauge vitrectomy. The 27-gauge EVA flow mode produced optimal intraoperative intraocular pressure stability.

CTLA-2 Alpha Is a Potent Inhibitor of Angiogenesis in Murine Ocular Tissue.

Cytotoxic T lymphocyte antigen-2 (CTLA-2) alpha has been reported to suppress the activities of cathepsin L (Cath L), which is deeply involved in angiogenesis. Therefore, we assessed whether CTLA-2 alpha plays a role in angiogenesis in ocular tissue. To establish models of corneal inflammation and experimental choroidal neovascularization (CNV), male C57BL/6J mice (n = 5) underwent corneal suture placement or laser-induced CNV, respectively. Mice were then injected with recombinant CTLA-2 alpha (1 µg) into the peritoneal cavity at day 0 and every 2 days after operation. In vitro experiments were performed to assess the inflammatory response by measuring TNF-alpha secretion in peritoneal cavity exudate cells (PECs) or the proliferation of mouse vascular endothelial cells (mVECs). CTLA-2 alpha treatment dramatically suppressed corneal angiogenesis, as well as laser-induced CNV. Moreover, CTLA-2 alpha inhibited the proliferation of mVECs in vitro, while CTLA-2 alpha abolishment was able to rescue proliferation. However, CTLA-2 alpha could not suppress cytokine secretion from inflammatory cells such as PECs. In summary, CTLA-2 alpha was able to suppress angiogenesis by suppressing endothelial cell proliferation. Further studies are needed to investigate its usefulness as a new antiangiogenic treatment for a variety of conditions, including age-related macular degeneration.

Prospective Clinical Trial of Intravitreal Aflibercept Treat-and-extend Regimen for Diabetic Macular Edema: 1-Year Outcomes.

PURPOSE: To investigate the efficacy of aflibercept for the treatment of diabetic macular edema via a treat-and-extend regimen. METHODS: This prospective, single-center, open-label, interventional study involved 30 patients with a best-corrected visual acuity (BCVA) ≤0.6 and a central retinal thickness (CRT) ≥300 µm. The enrolled eyes each received a monthly intravitreal aflibercept injection until the CRT decreased below 300 µm, upon which the administration interval was extended for 1 month until the CRT once again increased to ≥300 µm. Main outcome measures were median BCVA and CRT at 6 and 12 months after initiation of treatment via last observation carried forward analysis, the median number of injections over the 12 months, and the effects on the diabetic retinopathy severity scale (DRSS) score of the patients who completed the 12-month follow-up period. RESULTS: Of the 30 enrolled patients, 29 and 25 respectively completed the 6- and 12-month follow-up examinations. From baseline to 6 and 12 months after treatment initiation, the median BCVA (logarithm of the minimum angle of resolution) significantly improved from 0.52 to 0.30 and 0.35, respectively, and the median CRT significantly decreased from 439.5 to 268.5 and 249.0 µm, respectively. The median number of injections over the 12-month follow-up period was 6.0. Compared to baseline, the DRSS score at 12 months was improved by 2 steps in 16% of patients; in no cases did the DRSS score worsen or improve by three steps or more. CONCLUSIONS: When administered in a treat-and-extend regimen, aflibercept is an effective treatment option for diabetic macular edema.

Evaluation of pre- and post-surgery reading ability in patients with epiretinal membrane: a prospective observational study.

BACKGROUND: This study aimed to investigate the pre- and post-surgery reading ability in patients with idiopathic epiretinal membrane (ERM) to evaluate whether measurement of reading performance is a helpful test in addition to visual acuity (VA) as an assessment measure. METHODS: This prospective observational study involved 42 eyes of 40 patients with idiopathic ERM. Best-corrected visual acuity (BCVA), reading ability, and metamorphopsia score were evaluated at baseline and at 3, 6, and 12 months post-surgery. As the outcome measure, the reading ability of each patient (i.e., overall performance) was examined with MNREAD-J, the Japanese version of the MNREAD reading acuity (RA) charts, to determine RA, critical print size (CPS), and maximum reading speed (MRS). Generally, a difference of 0.2 logMAR or more is considered a significant change in BCVA. Thus, as a subgroup analysis, we additionally evaluated the BCVA and reading ability of the patients with a BCVA difference of 0.1 logMAR or less between at baseline and at 12 months post-surgery. RESULTS: Relative to their values at baseline, the subjects exhibited significantly improved BCVA, RA, and CPS throughout the post-surgery examination period (P < 0.001) and significantly improved MRS at 12 months post-surgery (P = 0.04). No significant change in the vertical metamorphopsia score was observed throughout the post-surgery follow-up period. However, and compared to the value at baseline, significant improvements in the horizontal metamorphopsia score were observed at 3, 6 (P < 0.05), and 12 months (P < 0.001) post-surgery. In the subgroup analysis of the 23 eyes that exhibited a BCVA improvement of 0.1 logMAR or less, the median BCVA did not change, but the median RA and CPS improved by 0.2 logMAR. CONCLUSIONS: Our findings showed that the surgical removal of ERM improves reading ability, even when the BCVA score does not improve. The measurement of reading performance appears to be a helpful test in addition to VA as a measure for assessing the surgical removal of ERM.

Short axial length and hyperopic refractive error are risk factors of central serous chorioretinopathy.

BACKGROUND/AIMS: To evaluate the axial length (AL) and refractive status in central serous chorioretinopathy (CSC). METHODS: This retrospective observational case series involved 140 patients with CSC (180 eyes) and 78 age-matched and gender-matched control subjects. A detailed ophthalmic examination was performed, including an interferometer measurement of AL. Multimodal imaging comprised colour fundus photography, fluorescein angiography, indocyanine green angiography, fundus autofluorescence photography and spectral domain optical coherence tomography. RESULTS: Eighty eyes of 40 patients were categorised into the bilateral-CSC (b-CSC) group and 100 eyes of 100 patients were categorised into the unilateral-CSC (u-CSC) group. AL of the b-CSC (23.19 mm) and u-CSC (23.75 mm) groups was significantly shorter than that of the control (24.85 mm) group (p<0.001 for both). Moreover, AL was significantly shorter in the b-CSC group than in the u-CSC group (p=0.020). Spherical equivalent (SE) in the b-CSC (0.25 D) group was significantly greater than in the u-CSC (-0.81 D) and control (-1.38 D) groups (p<0.001 for both). Gender (male; OR 4.55; 95% CI 1.13 to 18.40; p=0.033), AL (OR 0.38; 95% CI 0.23 to 0.63; p<0.001), area of choroidal vascular hyperpermeability (OR 1.08; 95% CI 1.03 to 1.13; p=0.002) and presence of descending tract (OR 7.22; 95% CI 1.86 to 28.00; p=0.004) were the variables found to be significantly associated with b-CSC via multiple regression analyses. CONCLUSION: Anatomical features, such as shorter AL and greater SE, may be associated with the pathogenesis of CSC.

Multicenter survey of sutureless 27-gauge vitrectomy for primary rhegmatogenous retinal detachment: a consecutive series of 410 cases.

PURPOSE: To evaluate the surgical outcomes of the 27-gauge (G) vitrectomy system for the treatment of primary rhegmatogenous retinal detachment (RRD). METHODS: This retrospective consecutive series multicenter study involved a total of 410 eyes of 406 patients who underwent 3-port transconjunctival 27G pars plana vitrectomy (PPV) for RRD between November 2014 and December 2016 and who were followed for a minimum of 3 months postoperative. The main outcome measure was primary reattachment, with the secondary outcome measures being final reattachment, improvement of visual acuity (VA), intraocular pressure (IOP), intraoperative and postoperative complications, and surgery time. RESULTS: Of the 410 treated eyes, primary reattachment was achieved in 392 (95.6%) and final reattachment was achieved in 410 (100%). In 226 eyes (55.1%) with macula-on RRD, the mean logarithm of the minimum angle of resolution (logMAR) VA improved from 0.16 ± 0.51 pre-surgery to 0.02 ± 0.14 post-surgery (P = 0.11). In 184 eyes (44.9%) with macula-off RRD, logMAR VA improved from 1.06 ± 0.77 pre-surgery to 0.26 ± 0.35 post-surgery (P < 0.001). Following surgery, the mean IOP was highest at 1 day (15.7 ± 7.0 mmHg) postoperative. In all eyes, surgery was concluded without the use of sutures or the need of conversion to a larger-gauge instrument. Although hypotony was observed in 14 (3.4%) of the 410 treated eyes at 1 day postoperative, it spontaneously resolved within 1 week without additional surgical intervention. No postoperative complications such as infectious endophthalmitis were observed throughout the follow-up period. CONCLUSION: Our findings show that 27G PPV is both safe and effective for the treatment of primary RRD.

pH balance and lactic acid increase in the vitreous body of diabetes mellitus patients.

Although there have been no previous reports on the pH of the human vitreous body, it has been highly theorized that it changes in patients with diabetes mellitus (DM). In humans, it is necessary to measure the vitreous pH in vitro, which is an important point that presents a major problem, as vitreous pH immediately changes when exposed to air. The purpose of this present study was to report our recent development of an in vitro method for measuring vitreous pH via the combination of 27-gauge (G) vitreous surgery and a blood gas analyzer, as well as our investigative findings on whether or not there is a difference of pH depending on the presence of diabetes mellitus (DM). This cross-sectional study involved 30 subjects [18 subjects without DM (DM-) and 12 subjects with DM (DM+)] with no previous history of ophthalmologic surgery. The DM+ group included 6 cases of proliferative diabetic retinopathy (PDR) and 6 cases of non-PDR (NPDR). The DM- Group was comprised of patients with a macular hole or idiopathic epiretinal membrane. The DM+ Group included patients not only with macular hole or idiopathic epiretinal membrane but also diabetic macular edema, however, patients with obvious vitreous hemorrhage were excluded. In all patients, a vitreous specimen was anaerobically obtained at the start of 27G pars plana vitrectomy, with a venous blood sample being collected immediately prior to surgery. Between the DM- and DM+ subjects, pH, partial pressure of carbon dioxide (pCO2), partial pressure of oxygen (pO2), K+, Na+, Ca2+, Cl-, lactate, and glucose were compared. In the items in which a significant difference was found between DM- and DM+, the values between the PDR and NPDR cases were also compared. Our findings showed no significant difference in vitreous and venous-blood pH between the DM- and DM+ subjects. The vitreous biochemical data revealed that Ca2+ significantly reduced and lactate and glucose significantly increased in DM+ compared to DM-. Thus, we compared Ca2+, lactate, and glucose between the PDR and NPDR cases. Although glucose did not significantly change, Ca2+ significantly decreased and lactate significantly increased in the PDR cases. The venous biochemical data revealed that only glucose significantly increased in DM+. The data in all investigated items was found to be significantly different between the vitreous and venous samples. Our findings revealed that lactate increases and Ca2+ decreases in the vitreous body of DM patients, especially those with PDR, probably due to the increased production of lactic acid. However, although the production of lactic acid increased, the pH remained at a nearly constant value, thus suggesting that the human vitreous body has a high buffering capacity.

The Efficacy of Sodium-Glucose Cotransporter 2 (SGLT2) inhibitors for the treatment of chronic diabetic macular oedema in vitrectomised eyes: a retrospective study.

OBJECTIVE: To investigate the change of chronic diabetic macular oedema (DMO) in vitrectomised eyes when the administration of sodium-glucose cotransporter 2 (SGLT2) inhibitors is initiated as a systemic medical treatment. METHODS AND ANALYSIS: This study involved 10 eyes of five patients with chronic DMO lasting more than 6 months who had previously undergone vitrectomy and whose systemic medical treatments were newly changed to SGLT2 inhibitors. In this study, chronic DMO was defined as persistent diffuse macular oedema despite ophthalmic treatment in patients with diabetes. Patients who received antivascular endothelial growth factor therapy or steroids administration, or change of eye-drop medication from at 3 months before and after the initiation of SGLT2 inhibitors, were excluded. In this study, visual acuity (VA) and central retinal thickness (CRT, µm) prior to and at 3, 6 and 12 months after the initiation of SGLT2 inhibitors were retrospectively compared. The Wilcoxon signed-rank test was used for statistical analysis. RESULTS: In the 10 treated eyes, from at baseline to at 3, 6 and 12 months after the initiation of SGLT2 inhibitor, median VA (logMAR) improved from 0.35 to 0.15 (p=0.038), 0.2 (p=0.157) and 0.2 (p=0.096), respectively, and median CRT significantly reduced from 500.5 µm to 410 µm (p<0.01), 378 µm (p<0.01) and 339 µm (p<0.01), respectively. CONCLUSION: Although this study involved only five patients, our findings indicate that SGLT2 inhibitors might have structural efficacy for chronic DMO in vitrectomised eyes.

Immunohistochemical Detection of Propionibacterium acnes in the Retinal Granulomas in Patients with Ocular Sarcoidosis.

The etiology of sarcoidosis is still obscure; however, Mycobacteria and Propionibacterium acnes are considered the most implicated etiological agent for sarcoidosis. To investigate whether P. acnes is an etiological agent for sarcoid uveitis, we analyzed the frequency of P. acnes detected within the biopsied retinas from patients with ocular sarcoidosis by immunohistochemistry with a P. acnes-specific monoclonal antibody (PAB antibody). Eleven patients (12 eyes) with sarcoid uveitis were enrolled in this study. Eight patients with rhegmatogenous retinal detachment, two patients with non-sarcoid uveitis, and two patients with vitreoretinal lymphoma were enrolled as controls. In the sarcoidosis group, granulomas were mainly observed in the inner retinal layer filled with CD4+ cells and CD68+ cells, indicating the Th1 immune response. P. acnes, identified as round bodies that reacted with the PAB antibody, were present in 10/12 samples (83%) from 9/11 patients (82%) with sarcoidosis. These round bodies were scattered within the retinal granulomas mainly in the inner retinal layer. In the control group, no round bodies were detected. Our results suggested that P. acnes could be associated with sarcoid uveitis. We hypothesize that sarcoid granulomas may be formed by a Th1 immune response to P. acnes hematogenously transmitted to the retina.

SURGICAL OUTCOMES OF 27-GAUGE VITRECTOMY FOR A CONSECUTIVE SERIES OF 163 EYES WITH VARIOUS VITREOUS DISEASES.

PURPOSE: To evaluate the safety and efficacy of 27-gauge vitrectomy for various vitreoretinal disorders. METHODS: In this retrospective comparative study, 163 consecutive eyes with various diseases that underwent 27-gauge pars plana vitrectomy with or without ultraspeed transformer by a single surgeon from June 2012 through December 2014 were analyzed in regard to best-corrected visual acuity, intraocular pressure, intraoperative and postoperative complications, and surgery time. RESULTS: In 2 eyes (1.2%), peripheral retina breaks were encountered intraoperatively, yet no other complications were found in those eyes. No cases required larger-gauge vitrectomy. Mean best-corrected visual acuity improved from 20/58 (logarithm of the minimum angle of resolution, 0.46 ± 0.64) preoperatively to 20/32 (logarithm of the minimum angle of resolution, 0.20 ± 0.40) postoperatively (P < 0.001). Mean follow-up was 16.7 months (range, 6-33 months). Intraocular pressure remained stable throughout the postoperative course. Hypotony was seen in 15 eyes (9.2%) at 1-day postoperative, yet that spontaneously improved within 1 week. No case of retinal detachment or endophthalmitis was recorded. In macular surgeries, such as idiopathic epiretinal membrane and macular hole combined with cataract surgery, the mean surgery time was 32.1 ± 6.9 minutes with ultraspeed transformer (n = 38) and 37.1 ± 7.7 minutes without ultraspeed transformer (n = 40) (P = 0.004). CONCLUSION: The 27-gauge pars plana vitrectomy was found to be safe and effective for treating various vitreoretinal disorders.

A case of fungal keratitis and endophthalmitis post penetrating keratoplasty resulting from fungal contamination of the donor cornea.

PURPOSE: Fungal infections post keratoplasty due to contamination of the donor corneal graft have become important issues that need to be addressed. Here we report a case of fungal keratitis and endophthalmitis post penetrating keratoplasty (PKP) due to fungal contamination of the donor corneal graft. OBSERVATIONS: We present a 52-year-old male who underwent PKP with a donor corneal graft that was later found to be contaminated with fungus. At 4-weeks postoperative, infectious infiltrates suddenly appeared at the border between the host and donor corneal graft, and endophthalmitis concomitantly occurred. A culture of the remnant donor corneoscleral rims and the vitreous fluid obtained during vitreous surgery was found to be positive for Candida albicans. At 6-months post vitreous surgery and intensive anti-fungal medical treatment, both corneal infiltrates and vitreous opacity completely disappeared, and the patient's best-corrected visual acuity recovered to 20/40, with a transparent cornea. CONCLUSIONS AND IMPORTANCE: The findings of this case show that prompt intensive medical treatment and surgical intervention effectively saved the vision in a patient with fungal keratitis and endophthalmitis due to contamination of the donor corneal graft.

Stage-specific reference genes significant for quantitative PCR during mouse retinal development.

Developing mouse retina has been serving as an ideal model for investigating the molecular mechanism of neural development and angiogenesis, because several significant events associated with these physiological phenomena are drastically occurring in conjunction with retinal development. However, as many genes are influencing on each other to establish mature retina within 21 days from E10 to P12, we must carefully design the experiments, such as in the case of quantitating the amount of altered gene expression toward the establishment of retina by quantitative PCR. As we have seen considerable variations of quantitative results in different developmental stages of retina depending on the reference genes used for compensation, we here attempted to determine a reliable reference gene to accurately quantitate the target genes in each stage. According to the results of in silico prediction and comparison with a database of SAGE, we found that the most stable gene from early to late stages was Sdha, whereas one of the most popular housekeeping genes, Actb, was the one that could mislead the quantitative results even in the adult stage. Consequently, we pointed out the importance of selecting an appropriate reference gene, especially to quantitate the amount of gene expression in the developmental stages of a certain tissue.

The CD4/CD8 ratio in vitreous fluid is of high diagnostic value in sarcoidosis.

PURPOSE: Sarcoidosis is an idiopathic inflammatory disorder involving multiple organs, and ocular manifestation (represented by granulomatous uveitis) is one of the common features. A well-known immunologic feature in sarcoidosis is an increased CD4+ helper T-cell type 1 lymphocyte subset in bronchoalveolar lavage (BAL) fluid. The current study investigated the vitreous lymphocyte subsets of ocular sarcoidosis to elucidate the immunologic features of this disorder in the eye. DESIGN: Case-control study. PARTICIPANTS AND CONTROLS: Fifty-one eyes of 38 patients with ocular sarcoidosis, confirmed by international diagnostic criteria, were enrolled in this study. Twenty-seven eyes of 26 patients with other causes of uveitis were enrolled as nonsarcoid controls. METHODS: Evaluation of diagnostic tests for cell profiles of ocular sarcoidosis. Lymphocytes in the vitreous samples were analyzed by cytology, polymerase chain reaction, and flow cytometry. Peripheral blood was also obtained from each patient and analyzed in comparison with the vitreous samples. MAIN OUTCOME MEASURES: CD4/CD8 ratios of vitreal and peripheral T lymphocytes. RESULTS: CD4/CD8 ratios of the vitreous T lymphocytes were significantly higher in ocular sarcoidosis than in nonsarcoidosis vitreous samples. In the patients with ocular sarcoidosis, the CD4/CD8 ratios of vitreal T lymphocytes were significantly higher than the CD4/CD8 ratios of peripheral T lymphocytes. No significant differences were found between the CD4/CD8 ratios of vitreal and peripheral T lymphocytes in the patients without sarcoidosis. Moreover, the CD4/CD8 ratios of peripheral T lymphocytes in the patients with ocular sarcoidosis were significantly higher than in patients without sarcoidosis. The sensitivity and specificity of the vitreal CD4/CD8 ratio were 100% and 96.3%, respectively, for the diagnosis of ocular sarcoidosis. CONCLUSIONS: Our findings suggest that the CD4/CD8 ratio of vitreous-infiltrating lymphocytes has high diagnostic value in ocular sarcoidosis, comparable to that of the CD4/CD8 ratio in BAL fluid lymphocytosis for pulmonary sarcoidosis. Furthermore, a high CD4/CD8 ratio of peripheral blood T lymphocytes should be one of the laboratory findings for ocular sarcoidosis. Diagnostic vitrectomy using flow cytometric analysis may be a useful adjunct for the diagnosis of ocular sarcoidosis, particularly in complex cases.

Simultaneous analysis of multiple cytokines in the vitreous of patients with sarcoid uveitis.

PURPOSE: Levels of some cytokines are significantly higher in the vitreous fluid of patients with acute uveitis than in normal vitreous fluid. The authors sought to determine which proinflammatory cytokines were upregulated in the vitreous fluid of patients with ocular sarcoidosis. METHODS: Samples of vitreous fluid were collected from patients with sarcoid uveitis and from nonsarcoid control patients with idiopathic epiretinal membrane. The levels of 27 proinflammatory cytokines were measured with a multiplex beads array system. Postvitrectomy macular thickness was also measured by using spectral domain optical coherence tomography (SD-OCT). To assess the relationship between cytokine levels and disease stage, the authors divided patients into three groups based on macular thickness 1 month after operation. RESULTS: The vitreous levels of 17 cytokines were significantly higher in patients with ocular sarcoidosis than in nonsarcoid controls. Serum levels of interferon γ-induced protein 10 (IP-10) were also higher in ocular sarcoidosis patients than in nonsarcoid controls. Conversely, serum levels of interleukin (IL) 15 in ocular sarcoidosis patients were lower than in the control group. Analysis of cytokine levels and macular thickness revealed that IL-1ra, IL-4, IL-8, IFN-γ, IP-10, monocyte chemotactic protein (MCP)-1, macrophage inflammatory protein (MIP)-1ß, and regulated on activation, normal T-cell expressed and secreted (RANTES) were significantly upregulated in patients with thin cystoid macular edema group. CONCLUSIONS: Patients with ocular sarcoidosis had elevated levels of proinflammatory cytokines in vitreous fluids. Different cytokines might contribute to different stages of macular edema.

Heat treatment of retinal pigment epithelium induces production of elastic lamina components and antiangiogenic activity.

Age-related macular degeneration (AMD) is the leading cause of blindness in the Western world. In advanced AMD, new vessels from choriocapillaris (CC) invade through the Bruch's membrane (BrM) into the retina, forming choroidal neovascularization (CNV). BrM, an elastic lamina that is located between the retinal pigment epithelium (RPE) and CC, is thought to act as a physical and functional barrier against CNV. The BrM of patients with early AMD are characterized by decreased levels of antiangiogenic factors, including endostatin, thrombospondin-1 (TSP-1), and pigment epithelium-derived factor (PEDF), as well as by degeneration of the elastic layer. Motivated by a previous report that heat increases elastin expression in human skin, we examined the effect of heat on human ARPE-19 cell production of BrM components. Heat treatment stimulated the production of BrM components, including TSP-1, PEDF, and tropoelastin in vitro and increased the antiangiogenic activity of RPE measured in a mouse corneal pocket assay. The effect of heat on experimental CNV was investigated by pretreating the retina with heat via infrared diode laser prior to the induction of CNV. Heat treatment blocked the development of experimental CNV in vivo. These findings suggest that heat treatment may restore BrM integrity and barrier function against new vessel growth.

Disease-related quantitation of TGF-beta3 in human aqueous humor.

TGF-beta3 has been implicated in the pathology of ocular diseases, but its concentration in human aqueous humor has never been assessed. In this study, we established an enzyme-linked immunosorbent assay (ELISA) for TGF-beta3 and quantitated it in aqueous humor collected from patients with pseudoexfoliation syndrome (PE), primary open angle glaucoma, chronic angle closure glaucoma and cataract (as the control). To develop the TGF-beta3 ELISA, we screened antibodies to identify the best combination, validated the assay for aqueous humor, and optimized the procedure for preparing activated TGF-beta3. As a result, our ELISA was highly selective and reproducible. Using our ELISA, we discovered a significantly elevated concentration of TGF-beta3 in PE eyes. We also developed new TGF-beta1 and -beta2 ELISAs, and were able, for the first time, to quantitate all the TGF-beta isoforms in the aqueous humor from a single eye, to assess their proportional effects on the pathogenesis of ocular diseases.

Oxidized phospholipids in the macula increase with age and in eyes with age-related macular degeneration.

PURPOSE: There is good evidence that oxidative stress is involved in the pathogenesis of age-related macular degeneration (AMD). Because AMD has risk factors and histopathology similar to with atherosclerosis, we hypothesized that oxidized phospholipids, which contribute to the pathogenesis of atherosclerosis, would accumulate in the eyes of AMD patients. To test this hypothesis, we investigated whether oxidized phospholipids were present in normal eyes and whether the level changed with increasing age. We then, we determined whether the levels of oxidized phospholipids were higher in eyes with AMD. METHODS: Twenty normal human donor eyes and six eyes with AMD were studied. Immunohistochemistry was performed on a tissue strip from the macular region using an antibody against oxidized phosphatidylcholine. Western blot analysis was also performed on proteins extracted from the posterior retina of donor eyes. The immunoreactivity of the specimens and the bands were quantified with NIH image software. RESULTS: Immunohistochemistry showed oxidized phosphatidylcholine was present in the photoreceptors and retinal pigment epithelium of the normal human macular area, and their levels increased with age. Eyes with AMD showed more intense immunoreactivity for oxidized phospholipids than age-matched normal eyes. CONCLUSIONS: These findings suggest that oxidative stress is involved in the pathogenesis of AMD possibly by oxidizing phospholipids in the photoreceptors as demonstrated in the arterial intima of patients with atherosclerosis. It is likely that controlling oxidation of phospholipids may be a potential treatment for AMD.

Scavenger receptors for oxidized lipoprotein in age-related macular degeneration.

PURPOSE: The accumulation of macrophages is known to be involved in the pathogenesis of age-related macular degeneration (AMD), but the reasons why macrophages accumulate in AMD lesions have not been determined. Because the histopathology of AMD has some factors common with those of atherosclerosis, the authors hypothesized that macrophages accumulate to take up oxidized lipoproteins in the eyes of patients with AMD, as has been demonstrated in atherosclerosis. METHODS: Immunohistochemistry was performed on 10 surgically excised choroidal neovascular (CNV) membranes from eyes with AMD. An antibody against oxidized lipoprotein and antibodies against the scavenger receptors SR-PSOX and LOX-1 were used. Antibodies against cytokeratin, CD68, and von Willebrand factor were used to identify retinal pigment epithelium (RPE), macrophages, and vascular endothelial cells, respectively. RT-PCR was performed to detect the mRNAs of the scavenger receptors in the CNV membranes. RESULTS: Oxidized lipoproteins were immunohistochemically detected in the CNV membranes. Intense immunostaining was observed at the surface of the CNV membranes with the SR-PSOX antibody, whereas LOX-1 immunostaining was weak. Cells expressing scavenger receptors were found to be predominantly macrophages with a minority of RPE. Both SR-PSOX and LOX-1 mRNAs were detected in CNV membranes. CONCLUSIONS: Oxidized lipoproteins are present in AMD lesions. Macrophages and RPE in the CNV membranes express cell surface scavenger receptors for oxidized lipoproteins. These findings suggest that macrophages may accumulate to take up oxidized lipoproteins in AMD and that the control of oxidative stress and macrophage responses may therefore be potential treatments for AMD.