Hsiao-Ming Chao

Resveratrol Mitigates Rat Retinal Ischemic Injury: The Roles of Matrix Metalloproteinase-9, Inducible Nitric Oxide, and Heme Oxygenase-1

PURPOSE Retinal ischemia-associated ocular disorders, such as retinal occlusive disorders, neovascular age-related macular degeneration, proliferative diabetic retinopathy, and glaucoma are vision-threatening. In this study, we examined whether and by what mechanisms resveratrol, a polyphenol found in red wine, is able to protect against retinal ischemia/reperfusion injury. METHODS In vivo rat retinal ischemia was induced by high intraocular pressure (HIOP), namely, 120 mmHg for 60 min. The mechanism and management was evaluated by electroretinogram (ERG) b-wave amplitudes measurement, immunohistochemistry, and real-time polymerase chain reaction. RESULTS The HIOP-induced retinal ischemic changes were characterized by a decrease in ERG b-wave amplitudes, a loss of choline acetyltransferase immunolabeling of amacrine cell bodies/neuronal processes, and increased vimentin immunoreactivity, which is a marker of Müller cells, together with upregulation of matrix metalloproteinase-9 (MMP-9), heme oxygenase-1 (HO-1), and inducible nitric oxide (iNOS), and downregulation of Thy-1, both at the mRNA level. The detrimental effects due to the ischemia were concentration-dependent (weaker effect at 0.05 nmole) and/or significantly (at 0.5 nmole) altered when resveratrol was applied 15 min before or after retina ischemia. CONCLUSION This study supports the hypothesis that resveratrol may be able to protect the retina against ischemia by downregulation of MMP-9 and iNOS, and upregulation of HO-1.

Baicalein Protects Against Retinal Ischemia by Antioxidation, Antiapoptosis, Downregulation of HIF-1α, VEGF, and MMP-9 and Upregulation of HO-1

PURPOSE Retinal ischemia-associated ocular disorders are vision threatening. This study examined whether the flavonoid baicalein is able to protect against retinal ischemia/reperfusion. METHODS Using rats, the intraocular pressure was raised to 120 mmHg for 60 min to induce retinal ischemia. In vitro, an ischemic-like insult, namely oxidative stress, was established by incubating dissociated retinal cells with 100 μM ascorbate and 5 μM FeSO4 (iron) for 1 h. The rats or the dissociated cells had been pretreated with baicalein (in vivo: 0.05 or 0.5 nmol; in vitro: 100 μM), vehicle (1% ethanol), or trolox (in vivo: 5 nmol; in vitro: 100 μM or 1 mM). The effects of these treatments on the retina or the retinal cells were evaluated by electrophysiology, immunohistochemistry, terminal deoxynucleotidyl-transferase-mediated dUTP nick end-labeling (TUNEL) staining, Western blotting, or in vitro dichlorofluorescein assay. In addition, real-time-polymerase chain reaction was used to assess the retinal expression of hypoxia-inducible factor-1α (HIF-1α), matrix metalloproteinase-9 (MMP-9), vascular endothelium growth factor (VEGF), and heme oxygenase-1 (HO-1). RESULTS The retinal changes after ischemia included a decrease in the electroretinogram b-wave amplitude, a loss of choline acetyltransferase immunolabeling amacrine cell bodies/neuronal processes, an increase in vimentin immunoreactivity, which is a marker for Müller cells, an increase in apoptotic cells in the retinal ganglion cell layer linked to a decrease in the Bcl-2 protein, and changes in the mRNA levels of HIF-1α, VEGF, MMP-9, and HO-1. Of clinical importance, the ischemic detrimental effects were concentration dependently and/or significantly (0.05 nmol and/or 0.5 nmol) altered when baicalein was applied 15 min before retinal ischemia. Most of all, 0.5 nmol baicalein significantly reduced the upregulation of MMP-9; in contrast, 5 nmol trolox only had a weak attenuating effect. In dissociated retinal cells subjected to ascorbate/iron, there was an increase in the levels of reactive oxygen species, which had been significantly attenuated by 100 μM baicalein and trolox (100 μM or 1 mM; a stronger antioxidative effect at 1 mM). CONCLUSIONS Baicalein would seem to protect against retinal ischemia via antioxidation, antiapoptosis, upregulation of HO-1, and downregulation of HIF-1α, VEGF, and MMP-9. The antioxidative effect of baicalein would appear to play a minor role in downregulation of MMP-9.

Effects of epigallocatechin-3-gallate on rat retinal ganglion cells after optic nerve axotomy.

The purpose of this study was to investigate the effects of epigallocatechin-3-gallate (EGCG) in axotomized eyes and the pathways related to its action. Wistar rats received intracranial optic nerve (ON) axotomy 2 mm behind the globe in left eyes, whereas right eyes received sham operations. EGCG was administrated via intraperitoneal injection 30 min before and 4 days after axotomy. The density of retinal ganglion cell (RGC) was examined by a retrograde labeling technique. Western blot analysis was used to assess the expression of neuronal nitric oxide synthase (nNOS), Bax, Bcl-2, ERK and Akt. Optic nerve axotomy caused 54% RGC loss 7 days following surgery, and EGCG treatment reduced RGC loss by 12% (P = 0.017). The expression of the nNOS and pro-apoptotic Bax proteins were increased 5 days after axotomy, while EGCG treatment significantly blunted the up-regulation of the above two proteins (P = 0.04 and 0.02, respectively). Axotomy-induced p-ERK 1/2 and p-Akt proteins expression 5 days and 3 days following injury, respectively. Treatment with EGCG further enhanced p-ERK 1/2 and p-Akt expressions after axotomy. Inhibition of ERK and Akt pathways attenuated the protection of EGCG on RGC against axotomy damage. Thus, we demonstrated that administration of EGCG prior to axotomy promotes RGC survival. The neuroprotective capacity of EGCG appears to act through mediating nitric oxide, anti-apoptotic, and cell survival signaling pathways.

Baicalein Significantly Protects Human Retinal Pigment Epithelium Cells Against H2O2-Induced Oxidative Stress by Scavenging Reactive Oxygen Species and Downregulating the Expression of Matrix Metalloproteinase-9 and Vascular Endothelial Growth Factor

PURPOSE Age-related macular degeneration is a leading cause of blindness in the elderly. At a later stage, neovascular or exudative age-related macular degeneration can lead to severe central vision loss that is related to aging-associated cumulative oxidative stress of the human retinal pigment epithelium (hRPE) cells. Early prevention with antioxidants is mandatory. The aim of this study was to determine whether and how baicalein can act as an antioxidant. METHODS The methods used included lactate dehydrogenase, 2′,7′-dichloro-fluorescein diacetate, or enzyme-linked immunosorbent assay to measure cell viability, oxygen free radical levels, or the levels of vascular endothelial growth factor (VEGF)/matrix metalloproteinase-9 (MMP-9), respectively. RESULTS H₂O₂ dose-dependently reduced the cell viability of hRPE cells. This negative effect was dose-dependently (with a lower effect at 20µM) and significantly counteracted by pretreatment with baicalein (50µM). Treatment with H₂O₂ significantly stimulated the formation of oxygen free radicals. This increase was dose-dependently and significantly blunted by baicalein. Further, treatment with a sublethal dose of H₂O₂ was associated with an upregulation in the levels of VEGF and MMP-9. The increases in these proteins were also dose-dependently (with a lower effect at 20µM) and significantly (50µM) blunted by pretreatment with baicalein. CONCLUSION This study supports an antioxidative role for baicalein whereby it protects hRPE cells against H₂O₂-induced oxidative stress by downregulating the levels of VEGF and MMP-9, which are increased by H₂O₂.

The effects and underlying mechanisms of S-allyl l-cysteine treatment of the retina after ischemia/reperfusion.

PURPOSE Retinal ischemia-associated ocular disorders are vision-threatening. The aim of the present study was to examine whether S-allyl l-cysteine (SAC) is able to protect against retina ischemia/reperfusion injury. METHODS In vivo, retinal ischemia in the rat was induced by raising intraocular pressure (IOP) to 120 mmHg for 60 min. In vitro, an ischemic-like insult, namely oxidative stress, was established by incubating retinal ganglion cell-5 (RGC-5) with 500 μM H(2)O(2) for 24 h. The mechanisms involved in these processes were evaluated by electrophysiology, immunohistochemistry, and molecular biological approaches. RESULTS The retinal changes caused by the high IOP were characterized by a decrease in electroretinogram b-wave amplitudes, a loss of choline acetyltransferase immunolabeling amacrine cell bodies/neuronal processes, and an upregulation of the mRNA levels of hypoxia-inducible factor-1α (HIF-1α), vascular endothelium growth factor (VEGF), and matrix metalloproteinase-9 (MMP-9). The increased protein levels of HIF-1α, VEGF, and MMP-9 were also seen in RGC-5 cells subjected to defined oxidative stress. Of clinical importance, the ischemic/ischemic-like detrimental effects were concentration-dependently (least effect at 25 μM) and/or significantly (50 and/or 100 μM) blunted when SAC was applied 15 min before retinal ischemia or ischemic-like insult, respectively. CONCLUSION SAC would seem to protect against retinal ischemia by acting as an antioxidant and inhibiting the upregulation of HIF-1α, VEGF, and MMP-9.

Iron-generated hydroxyl radicals kill retinal cells in vivo : effect of ferulic acid

Siderosis bulbi is vision threatening. An investigation into its mechanisms and management is crucial. Experimental siderosis was established by intravitreous administration of an iron particle (chronic) or FeSO4 (acute). After siderosis, there was a significant dose-responsive reduction in eletroretinogram (a/b-wave) amplitude, and an increase in • OH level, greater when caused by 24 mM FeSO4 than that by 8 mM FeSO4. Furthermore, the FeSO4-induced oxidative stress was significantly blunted by 100 μM ferulic acid (FA). Siderosis also resulted in an excessive glutamate release, increased [Ca++]i, and enhanced superoxide dismutase immunoreactivity. The latter finding was consistent with the Western blot result. Obvious disorganization including loss of photoreceptor outer segments and cholinergic amacrines together with a wide-spreading ferric distribution across the retina was present, which were related to the eletro-retinographic and pathologic dysfunctions. Furthermore, b-wave reduction and amacrine damage were respectively, significantly, dose-dependently, and clearly ameliorated by FA. Thus, siderosis stimulates oxidative stress, and possibly, subsequent excitotoxicity, and calcium influx, which explains why the retina is impaired electro-physiologically and pathologically. Importantly, FA protects iron toxicity perhaps by acting as a free radical scavenger. This provides an approach to the study and treatment of the iron-related disorders such as retained intraocular iron and Alzheimer disease.

Therapeutic Effects and Mechanisms of Action of Mannitol During H2O2-Induced Oxidative Stress in Human Retinal Pigment Epithelium Cells

BACKGROUND Age-related macular degeneration (AMD) is a leading cause of blindness in the elderly. At a later stage, neovascular or exudative AMD can lead to severe central vision loss that is related to aging-associated cumulative oxidative stress of the human retinal pigment epithelium (hRPE) and choroid capillary. Early prevention with antioxidants is mandatory. The aim of this study was to determine whether and how mannitol can act as an antioxidant. METHODS The methods used included measurements of cell viability, oxygen free radical (OFR) levels, lipid peroxide (LP) levels, and OFR-related enzyme protein levels. RESULTS H(2)O(2) dose-dependently reduced the cell viability of hRPE cells. This negative effect was significantly counteracted by pretreatment with mannitol (1 mM). H(2)O(2) significantly stimulated the formation of OFR and LP. These increases were dose-dependently and significantly blunted by mannitol. Furthermore, treatment with H(2)O(2) was associated with a reduction in the level of catalase, but not of manganese superoxide dismutase (MnSOD). In contrast, it was shown that mannitol protected hRPE cells against the H(2)O(2)-induced oxidative stress by increasing the level of catalase, but not the level of MnSOD. CONCLUSION This study supports an antioxidative role for mannitol that acts through up-regulating the level of catalase, which is decreased by H(2)O(2).

Haploinsufficiency of RCBTB1 is Associated with Coats Disease and Familial Exudative Vitreoretinopathy

Familial exudative vitreoretinopathy (FEVR) belongs to a group of genetically and clinically heterogeneous disorders in retinal vascular development. To date, in approximately 50% of patients with FEVR, pathogenic mutations have been detected in FZD4, LRP5, TSPAN12, NDP and ZNF408. In this study, we identified two heterozygous frameshift mutations in RCBTB1 from three Taiwanese cases through exome sequencing. In patient-derived lymphoblastoid cell lines (LCLs), the protein level of RCBTB1 is approximately half that of unaffected control LCLs, which is indicative of a haploinsufficiency mechanism. By employing transient transfection and reporter assays for the transcriptional activity of β-catenin, we demonstrated that RCBTB1 participates in the Norrin/FZD4 signaling pathway and that knockdown of RCBTB1 by shRNA significantly reduced nuclear accumulation of β-catenin under Norrin and Wnt3a treatments. Furthermore, transgenic fli1:EGFP zebrafish with rcbtb1 knockdown exhibited anomalies in intersegmental and intraocular vessels. These results strongly support that reduced RCBTB1 expression may lead to defects in angiogenesis through the Norrin-dependent Wnt pathway, and that RCBTB1 is a putative genetic cause of vitreoretinopathies.

S-allyl L-cysteine protects the retina against kainate excitotoxicity in the rat.

Excitotoxicity has been proposed to play a pivotal role in retinal ischemia. Retinal ischemia-associated ocular disorders are vision threatening. The aim was to also examine whether and how S-allyl L-cysteine (SAC) can protect the retina against kainate excitotoxicity. In vivo retinal excitotoxicity was induced by an intravitreous injection of 100 μM kainate into a Wistar rat eye for 1 day. The management and mechanisms involved in the processes were evaluated by electrophysiology, immunohistochemistry, histopathology, and various biochemical approaches. In the present study, the cultured retinal cells were shown to possess kainate receptors. The defined retinal excitotoxic changes were characterized by a decrease in electroretinogram (ERG) b-wave amplitudes, a loss of the fluorogold retrograde labeled retinal ganglion cells (RGCs), an increase in the apoptotic cells in the RGC layer, and an increase in vimentin or glial fibrillary acidic protein (GFAP) immunoreactivity, a marker for Müller cells. An up-regulation in the mRNA levels of inducible nitric oxide synthase (iNOS) and matrix metalloproteinases-9 (MMPs-9) was also detected in the retina subjected to kainate excitoxicity. Importantly, the excitotoxicity-induced alterations were significantly blunted when 100 μM SAC and/or the kainate receptor antagonist CNQX was applied. Conclusively, SAC would seem to protect the retina against kainate excitotoxicity via an inhibition of the up-regulation of iNOS and MMP-9 as well as a modulation of glial activation and apoptosis.

Characterization and functional correlation of multiple imaging modalities with focal choroidal excavation

Background: To investigate the clinical manifestations and imaging features of near‐infrared autofluorescence (NIA), infrared reflectance (IR), fundus autofluorescence (FAF), indocyanine green angiography (ICGA) and fluorescein angiography (FAG) in the detection of patients with focal choroidal excavation (FCE) identified by cross‐sectional spectral‐domain optical coherence tomography (SD‐OCT). Methods: This retrospective cross‐sectional study included 12 eyes of 10 Taiwanese patients with FCE diagnosed by SD‐OCT. The areas and depths of FCE in serial cross‐sectional and en‐face OCT were compared in different imaging modalities. NIA, IR, FAF, ICGA and FAG images were obtained. Best corrected visual acuity, subjective distortion area in the Amsler grid and history of maculopathies were also recorded. Results: In areas where the choroid started to excavate as shown in SD‐OCT, hypo‐autofluorescence in NIA was noted. The area of hypo‐fluorescence in NIA of all the FCE lesions showed good correlation with the size. The area of FCE was associated with complications such as choroidal neovascularization and central serous chorioretinopathy (p = 0.014, d.f = 1) and the volume (NIA area × Depth measured by SD‐OCT × 1/3) was associated with subjective distortion strongly (p = 0.051, Spearmans correlation = 0.600). Conclusion: Among all image modalities, NIA was the most sensitive tool in area measurement of FCE and peripheral lesion detection. Also, the volume of FCE was associated with subjective distortion and the area was related to complications. Recording the area and volume of FCE could play an important role in monitoring complications.