Yanling Wang

Role of RhoA/MERK1/ERK1/2/iNOS signaling in ocular ischemic syndrome

PurposeTo determine the molecular mechanisms underlying ocular ischemic syndrome (OIS). This study uses a rat model to evaluate the role of the RhoA/MEK1/ ERK1/2/iNOS pathways in response to OIS-associated oxidative and nitrosative stress, with a long-term goal of identifying therapeutic targets for OIS.MethodsRats were randomly allocated to one of three groups: bilateral occlusion of the common carotid artery (BOCCA), sham surgery control, or unoperated control (n = 8/group). Three months after the procedure, retinas were analyzed anatomically, using immunohistochemistry and by enzyme-linked immunosorbent assay for RhoA, MEK1, ERK1, ERK2, iNOS. Retinal injury was assessed using TUNEL. Levels of superoxide dismutase (SOD) and malondialdehyde (MDA) were measured by WST-1 and TBA methods, respectively.ResultsIn BOCCA rats, occlusion of the bilateral common carotid artery induced degeneration of retinal ganglion cells, which was not observed in either control group. Retinal levels of RhoA, MEK1, ERK1, ERK2, iNOS, NOX2, and MDA were elevated in the BOCCA group, but not in either control group. In comparison, retinal levels of SOD were reduced in SOD animals. By immunofluorescent staining, RhoA was elevated in all retinal layers, while the increased levels of MEK, ERK1/1, and NOX were restricted to the INL, and that of ERK1/2 and NOX inner nuclear layer; iNOS elevations were observed in both the inner and outer nuclear layers. TUNEL labeling results showed that BOCCA group is higher staining than sham and control group.ConclusionsOIS elevates activity of the RhoA/MERK1/ERK1/2/iNOS pathways throughout the retina, likely reflecting a response to oxidative and nitrosative stress. Retinal thickness was reduced in BOCCA rats, reflecting a loss of retinal ganglion cells following the reduced blood flow to the eye. These results indicate that drugs that inhibit these pathways may be effective treatments for OIS.

TGF-β participates choroid neovascularization through Smad2/3-VEGF/TNF-α signaling in mice with Laser-induced wet age-related macular degeneration.

Choroidal neovascularization(CNV) is the most severe complication in Age-related macular degeneration(AMD) and the most common cause of irreversible blindness in the elderly in developed world. The aim of this study was to identify the effect of transforming growth factor-β(TGF-β) and Smad2/3-VEGF/TNF-α signaling on CNV angiopoiesis, and to explore TGF-β inhibitors on the development of CNV in a CNV mouse model. Fundus fluorescein angiography(FFA) was used to evaluate the laser-induced CNV formation. The histology of CNV lesions stained with hematoxylin-eosin(HE) was obtained. The immunofluorescent staining was performed to determine TGF-β protein expression. The expressions of TGF-β, phosphorylated Smad2/3, VEGF and TNF-α were determined by using Western blot analysis. The CNV areas were analyzed by using fluorescein stain on RPE/choroid-sclera flat mounts. We found the levels of TGF-β protein expression increasingly reached the peak till 3rd week during the CNV development. The protein levels of VEGF and TNF-α also increased significantly in CNV mice, which were inhibited by a synthetic TGF-β inhibitor LY2157299 or a natural TGF-β inhibitor Decorin. The phosphorylated Smad2/3 levels increased significantly in CNV mice, but this response was profoundly suppressed by the TGF-β inhibitors. Here we have demonstrated that TGF-β/Smad signaling plays an important role in Laser-induced CNV formation through down-regulation of VEGF and TNF-α expressions, suggesting TGF-β inhibitors may provide an alternative to traditional methods in wet AMD treatment.

α-Aminoadipic acid protects against retinal disruption through attenuating Müller cell gliosis in a rat model of acute ocular hypertension

Objective Ocular hypertension is an important risk factor for glaucoma. The purpose of this study was to investigate the gliotoxic effects of α-aminoadipic acid (AAA) in a rat model of AOH and its underlying mechanisms. Materials and methods In the rat model of acute ocular hypertension (AOH), intraocular pressure was increased to 110 mmHg for 60 minutes. Animals were divided into four groups: sham operation (Ctrl), AOH, AOH + phosphate-buffered saline (PBS), and AOH + AAA. Cell apoptosis in the ganglion cell layer was detected with the terminal deoxynucleotidyl transferase-mediated uridine 5′-triphosphate-biotin nick end labeling (TUNEL) assay, and retinal ganglion cells (RGCs) immunostained with Thy-1 were counted. Müller cell activation was detected using immunostaining with glutamine synthetase and glial fibrillary acidic protein. Tumor necrosis factor-α (TNF-α) was examined using Western blot. Results In the rat model of AOH, cell apoptosis was induced in the ganglion cell layer and the number of RGCs was decreased. Müller cell gliosis in the retinas of rats was induced, and retinal protein levels of TNF-α were increased. Intravitreal treatment of AAA versus PBS control attenuated these retinal abnormalities to show protective effects in the rat model of AOH. Conclusion In the retinas of the rat model of AOH, AAA treatment attenuated retinal apoptosis in the ganglion cell layer and preserved the number of RGCs, likely through the attenuation of Müller cell gliosis and suppression of TNF-α induction. Our observations suggest that AAA might be a potential therapeutic target in glaucoma.

Efficacy of Osthole in Management of Hypoperfused Retina

Purpose To determine the effect of osthole on the retina in a chronic cerebral hypoperfusion (CCH) rat model and to investigate its therapeutic activity. Methods Seventy-two rats were randomly allocated into 6 groups. CCH was induced by permanent bilateral common carotid artery occlusion (BCCAO) in five groups. Sham surgery was performed without occlusion of the artery in the sixth group (control group). Animals were administered with saline (model group), osthole (osthole-IG group), aspirin (aspirin group), or ginaton (ginaton group); the osthole-PI group was performed with peribulbar injection of osthole. Four rats in each group were sacrificed every 5 days after drug administration, and histopathology along with morphology of retina were observed. Fundus fluorescein angiography was performed before the animals were sacrificed at day 15. Retinal Akt, NF-κB, Bax, and Bcl-2 levels were assessed using immunohistochemistry, immunofluorescence, and reverse-transcription PCR; retinal injury was assessed using TUNEL in situ; retinal levels of superoxide dismutase (SOD) and malondialdehyde (MDA) were measured. Results Fundus fluorescein angiography revealed the retinal vascular diameter in the osthole-IG group rats to be wider than that in the model, osthole-PI, aspirin, or ginaton group rats. Histological analysis of retinal tissue revealed an increase in retinal thickness in all treatment groups, and significant improvement was noticed in the osthole-IG group. TUNEL staining revealed fewer apoptotic cells in the osthole-IG and osthole-PI groups than in the other groups. For immunohistochemistry results, in the osthole-IG group, levels of NF-κB and Akt were lower than those in the other treated groups, while levels of the ratio Bcl-2/Bax were higher. Levels of MDA were lower and levels of SOD were higher in the osthole-IG group than in the other groups. Conclusions Osthole protects the retina from ischemia injury secondary to CCH induced by BCCAO, mainly through anti-inflammatory, antioxidant, and antiapoptotic effects.

Is the Retinal Vasculature Related to β-Peripapillary Atrophy in Nonpathological High Myopia? An Optical Coherence Tomography Angiography Study in Chinese Adults

Purpose The association between β-peripapillary atrophy and the retinal vasculature in nonpathological high myopia is unclear. The aim of this study is to investigate whether β-peripapillary atrophy contribute to the changes of the retinal vasculature using optical coherence tomography angiography. Methods In a cross-sectional study, one hundred and thirty eyes with nonpathological high myopia were included. β-peripapillary atrophy was analysed using Image J software based on fundus photographs. A 3.0 × 3.0 mm2 grid and a 4.5 × 4.5 mm2 grid were used to scan parafoveal and peripapillary regions using optical coherence tomography angiography, respectively. Vessel density and fractal dimensions of the retina and foveal avascular zone were analysed and quantified using en face projection images. Correlations between the vascular density, foveal avascular zone, and β-peripapillary atrophy were determined. Results Using multivariate analysis, β-peripapillary atrophy was negatively correlated with the vessel density in radial peripapillary capillaries (p=0.002) even after adjusting for other variables. This relationship was also confirmed in the macula (superficial retinal plexus: p < 0.05; deep retinal plexus: p < 0.05). The vessel densities in the nasal and inferior sectors were more strongly correlated with β-peripapillary atrophy. Conclusions There was a negative correlation between β-peripapillary atrophy and the retinal vasculature in highly myopic eyes, especially in radial peripapillary capillaries and deep retinal plexus. β-peripapillary atrophy can be visualized and is a convenient structural feature that can benefit the early diagnosis and detection of chorioretinal atrophy in high myopia.

Multimodality Imaging Assessment of Ocular Ischemic Syndrome

Objectives To assess the underlying mechanisms of OIS and confirm the haemodynamic and retinal structure changes of early OIS. Methods An observational cross-sectional study was conducted of 60 internal carotid artery (ICA) stenosis patients, and they were divided into OIS and control group. Colour doppler imaging, optical coherence tomography, and fundus fluorescein angiography were performed. Results The middle cerebral artery (MCA) stenosis differs significantly between the two groups. More OIS patients had new collateral patency of posterior communicating artery (PCoA) and retrograde flow via the ophthalmic artery (OA) (p < 0.001). The peak systolic velocity (PSV) in central retinal artery (CRA) and choroidal thickness (CT) was significantly reduced in OIS patients (p = 0.001 and p < 0.001). The arm-retina time (ART) and the retinal arteriovenous passage time (AVP) were prolonged in OIS patients (p < 0.001 and p = 0.001). CT, ART, and PSV of the CRA showed high sensitivity, while ART and ICA stenosis grade showed high specificity for the diagnosis of OIS according to ROC curve. Conclusions Patients who suffered from severe ipsilateral ICA stenosis, new collateral patency of PCoAs, and MCA stenosis may be more susceptible to OIS. The most sensitive sign is PSV of CRA and CT, and the most specific sign is ART.

Ocular Image and Haemodynamic Features Associated with Different Gradings of Ipsilateral Internal Carotid Artery Stenosis

Objectives To analyse the changes of ocular haemodynamics and morphology in Chinese patients with internal carotid artery (ICA) stenosis in the current study. Methods A retrospective case-control study was conducted with 219 patients. The haemodynamic characteristics, the calibre of retinal vessels, and the subfoveal choroidal thickness (SFChT) were compared. We analysed the correlations with the degree of ipsilateral ICA stenosis. Results There were no significant differences among the groups in the central retinal artery equivalent (CRAE), central retinal vein equivalent (CRVE), and AVR (p = 0.073, p = 0.188, and p = 0.738, resp.). The peak systolic velocity (PSV) and end diastolic velocity (EDV) in the central retinal artery (CRA) and the posterior ciliary artery (PCA) were significantly lower than normal eyes (p < 0.001). The outer retinal layer thickness and SFChT values of the ICA stenosis groups were significantly lower than normal eyes (p = 0.030 and p < 0.001, resp.). Conclusion The PSV and EDV in CRA and PCA and the SFChT and outer retinal layer thickness of ICA eyes were significantly lower than normal eyes. ICA stenosis may impact choroidal haemodynamics, and decreased choroidal circulation might affect the discordance of the SFChT and the outer retinal layer thickness.

Collapsin Response Mediator Protein-2-induced Retinal Ischemic Injury in a Novel Mice Model of Ocular Ischemia Syndrome

Background: Collapsin response mediator protein-2 (CRMP2) has been shown to be involved in ischemia/hypoxia (IH) injury. We determined whether CRMP2 modulates ischemic injury in the retinal of Ocular ischemic syndrome (OIS). This study was to explore the molecular mechanisms underlying OIS in a novel mice model. Methods: Experiments were performed on adult male C57/BL6 mice that received bilateral internal carotid arteries ligation for 1, 2, or 4 weeks. The mice received injection of calpeptin group before occlusion for 4 weeks or not. The expression of CRMP2 in the retinal was examined by western blotting (WB) analysis and immunohistochemical analysis (IHC). The effects of ischemic injury on retinal were evaluated by fundus examination, fundus fluorescein angiography, electroretinogram, cell counting of retinal ganglion cell (RGC), and measurement of the thickness of the retina. Results: The veins dilated after chronic ischemia. In the electroretinography, the amplitudes of a- and b-waves kept diminishing in an ischemia time-dependent manner. Moreover, the tail vein-retinal circulation time prolonged in the 1- and 2-week group. In comparison, thickness of the retina decreased gradually with the ischemia time elapsed. WB analysis showed the CRMP2 and p-CRMP2 levels decreased in the 2- and 4-week groups. The results of IHC analysis were compatible with our results of WB. The loss of RGCs, decrease of the total reaction time and reduction of CRMP2 was alleviated by intravitreal injection of calpeptin. Conclusions: These results revealed that bilateral ligation of the internal carotid artery causes retinal ischemia in mice. Moreover, CRMP2 might play a pivotal role during the ischemic injury in the retina and inhibit the cleavage of CRMP2 can ameliorate the IH injury.