For the first time, a microscopic imaging assay for directly visualizing macromolecules leaked through the outer BRB in rodents was developed. Using this assay, the authors demonstrated the significance of outer BRB breakdown in diabetes and ischemia, which will have implications to the understanding, diagnosis, and treatment of diabetic macular edema and other ocular diseases with outer BRB defects. The microscopic imaging assay established in this study will likely be very useful to the development of drugs for macular edema.

Diabetic retinopathy (DR) is a major complication of diabetes and a leading cause of blindness in working-age Americans. DR is traditionally regarded as a disorder of blood-retina barriers, and the leakage of blood content is a major pathological characteristic of the disease. While the breakdown of the endothelial barrier in DR has been investigated extensively, the vascular leakage through the retinal pigment epithelium (RPE) barrier in the disease has not been widely acknowledged. As the blood content leaked through the RPE barrier causes excessive water influx to the retina, the breakdown of the RPE barrier is likely to play a causative role in the development of some forms of diabetic macular edema, a major cause of vision loss in DR. In this article, we will discuss the clinical evidences of the diabetes-induced RPE barrier breakdown, the alteration of the RPE in diabetes, the molecular and cellular mechanism of RPE barrier breakdown, and the research tools for the analysis of RPE barrier leakage. Finally, we will discuss the methodology and potential applications of our recently developed fluorescent microscopic imaging for the diabetes-or ischemia-induced RPE barrier breakdown in rodents.

Volume-sensitive outwardly rectifying (VSOR) Cl^- channels are activated during osmotic swelling and involved in the subsequent volume regulation in most animal cells. To test the hypothesis that the ClC-3 protein is the molecular entity corresponding to the VSOR Cl^- channel in cardiomyocytes, the properties of VSOR Cl^- currents in single ventricular myocytes isolated from ClC-3-deficient (Clcn3^-/-) mice were compared with those of the same currents in ClC-3-expressing wild-type (Clcn3^+/+) and heterozygous (Clcn3^+/-) mice. Basal whole-cell currents recorded under isotonic conditions in ClC-3-deficient and -expressing cells were indistinguishable. The biophysical and pharmacological properties of whole-cell VSOR Cl^- currents in ClC-3-deficient cells were identical in ClC-3-expressing cells. The VSOR Cl^- current density, which is an indicator of the plasmalemmal expression of functional channels, was essentially the same in cells isolated from these 3 types of mice and C57BL/6 mice. Activation of protein kinase C (PKC) by a phorbol ester was found to upregulate VSOR Cl^- currents in ClC-3-deficient and -expressing cardiomyocytes. This effect is opposite to the reported downregulatory effect of PKC activators on ClC-3-associated Cl^- currents. We thus conclude that functional expression of VSOR Cl^- channels in the plasma membrane of mouse cardiomyocytes is independent of the molecular expression of ClC-3.

Retinal neovascularization (NV) occurs in various ocular disorders including proliferative diabetic retinopathy, retinopathy of prematurity and secondary neovascular glaucoma, which often result in blindness. Vascular endothelial growth factor (VEGF) is an essential growth factor for angiogenesis, and is particularly regulated by hypoxia inducible factor-1alpha (HIF-1alpha) under hypoxic conditions. Therefore, HIF-1alpha and VEGF could provide targets for therapeutic intervention on retinal NV. In this study, we investigated the inhibitory effects of small interfering RNA (siRNA) targeting HIF-1alpha and VEGF on the expression of HIF-1alpha and VEGF in human umbilical vein endothelial cells (HUVEC) in vitro and on retinal NV in vivo. siRNA-expressing plasmids targeting human HIF-1alpha (HIF-1alpha siRNA) and human VEGF(165) (VEGF siRNA) were constructed. They were transfected and co-transfected to HUVEC and C57BL/6J mice of ischemic retinopathy model. HIF-1alpha siRNA and VEGF siRNA specifically downregulated HIF-1alpha and VEGF at both mRNA and protein levels in vitro and in vivo. Neovascular tufts and neovascular nuclei were decreased in gene therapy group compared to control hypoxia group. Co-transfection of HIF-1alpha siRNA and VEGF siRNA resulted in maximal effects on VEGF suppression in vitro and in vivo. It also manifested the maximal inhibitory effect on retinal NV. These results indicate that the application of HIF-1alpha siRNA and VEGF siRNA technology holds great potential as a novel therapeutic for retinal NV.

Background: To assess the diagnostic efficacy of optical coherence tomography (OCT) and OCT angiography (OCTA) in Parkinson's disease (PD).Methods: OCT was used to obtain macular parameters and peripapillary retinal nerve fiber layer (RNFL) thickness. The macular superficial retinal vessel and foveal avascular zone (FAZ) were quantified with OCTA.The area under the receiver operating characteristic curve (AUC) indicated the diagnostic efficacy of the parameters.Results: Thirty-five eyes from 35 PD patients and 35 eyes from 35 age-matched healthy subjects who served as controls were evaluated. The mean RNFL thickness overall and the thicknesses of the other three quadrants were similar in PD patients compared with controls (P≥0.358). The RNFL thickness at the temporal quadrant, total macular volume (TMV), macular retinal thickness (MRT), and ganglion cell-inner plexiform layer complex (GCL-IPL) thickness were reduced in the eyes of PD patients (P≤0.046). There was no difference between the CMT of PD patients compared with control subjects (P=0.163). The vessel length density (VLD) in the central, inner and full regions; vessel perfusion density (VPD) in all regions; and the FAZ circularity index in PD patients were significantly lower than in controls (P≤0.049). The AUC of the VLD in PD in the central, inner and full regions were 0.712, 0.728, and 0.650, respectively; The VPD in the central, inner and full region were 0.711, 0.756, and 0.682, respectively. The mean RNFL thickness in the temporal quadrant, TMV and MRT revealed an AUC of 0.718, 0.693 and 0.699, respectively. The VPD in the outer region, FAZ circularity and GCL-IPL thickness did not have diagnostic ability in distinguishing PD from normal eyes (P≥0.05). The AUCs of a combination of the VLD in the inner region and TMV, the VLD in the inner region and MRT, the VPD in the inner region and TMV, and the VPD in the inner region and MRT, were 0.843, 0.849, 0.849, and 0.848, respectively (P≤0.001).Conclusions: Decreased OCT and OCTA parameters were detected in the eyes of PD patients. Combined non-invasive measurements of OCT and OCTA had better diagnostic ability than either alone, and may provide an additional biomarker for PD progression.

It is controversial whether the ClC-3 protein, which is one of the voltage-dependent chloride channel ClC family members, is a candidate for the volume-sensitive outwardly rectifying (VSOR) Cl -channel per se or its regulator. Here, for the first time, we examined the single-channel properties of the VSOR Cl -channel in ventricular myocytes isolated from ClC-3-deficient mice. The single-channel current induced by cell swelling exhibited Cl -selectivity, mild outward rectification, and an intermediate unitary con-The volume-sensitive outwardly rectifying (VSOR) Cl-channel is ubiquitously expressed and plays an essential role in cell volume regulation in most animal cells [1][2][3]. It has recently been reported that this channel is involved in important cell functions such as proliferation [4] and apoptosis [5]. In spite of the importance of its functions, the molecular identification of this channel has not yet been achieved, although the ClC-3 protein, which is one of the members of the voltage-dependent chloride channel ClC family, has been considered as the candidate for the VSOR Cl Okazaki, ductance (around 38 pS). A Cl -channel blocker, 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS), reversibly inhibited the outward current. These single-channel properties were identical with those in ClC-3 expressing wild-type ventricular myocytes. These results indicate that the single-channel activity of the VSOR Cl -channel is independent of the expression of ClC-3 proteins in mouse ventricular myocytes. [The Japanese Journal of Physiology 55: [379][380][381][382][383] 2005]

RNAi targeting HIF-1alpha could inhibit the retinal neovascularization by approximately 65% through down-regulating the expression of HIF-1alpha and VEGF in the murine retinas, which may provide a powerful and novel therapeutic tool for ischemic-induced retinal diseases.

This study analyzed the optical coherence tomography angiography (OCTA) macular parameters in primary angle-closure glaucoma (PACG) patients after acute primary angle closure (APAC) episodes. Thirty-three patients with 33 APAC eyes and 33 primary angle closure suspect (PACS) eyes and 33 age-matched normal subjects (controls) were enrolled. Macular vessel density (VD) in central, inner, outer and full regions and foveal avascular zone (FAZ) parameters (area, perimeter and circularity index) were compared between APAC, PACS, and control eyes. For resolved APAC eyes, the VD in each macular region was significantly lower than that in control eyes, with less central and inner macular VD than PACS eyes. The central macular VD was significantly lower in PACS eyes than in controls. There was no difference in FAZ area and perimeter between APAC, PACS, and control eyes. FAZ circularity was highest in control eyes, followed by PACS eyes, and lowest in APAC eyes. The AUC, sensitivity and specificity of FAZ circularity were 0.944, 93.9% and 84.8%, respectively, in APAC eyes and 0.881, 84.8% and 81.8%, respectively, in PACS eyes. Therefore, FAZ circularity had the best discrimination capability for detecting both APAC and PACS eyes. Macular assessment with OCTA could provide an accurate early-stage diagnostic tool for PACG.