Hyun Beom Song

Impact insertion of transfer-molded microneedle for localized and minimally invasive ocular drug delivery.

It has been challenging for microneedles to deliver drugs effectively to thin tissues with little background support such as the cornea. Herein, we designed a microneedle pen system, a single microneedle with a spring-loaded microneedle applicator to provide impact insertion. To firmly attach solid microneedles with 140 μm in height at the end of macro-scale applicators, a transfer molding process was employed. The fabricated microneedle pens were then applied to mouse corneas. The microneedle pens successfully delivered rhodamine dye deep enough to reach the stromal layer of the cornea with small entry only about 1000 μm(2). When compared with syringes or 30 G needle tips, microneedle pens could achieve more localized and minimally invasive delivery without any chances of perforation. To investigate the efficacy of microneedle pens as a way of drug delivery, sunitinib malate proven to inhibit in vitro angiogenesis, was delivered to suture-induced angiogenesis model. When compared with delivery by a 30 G needle tip dipped with sunitinib malate, only delivery by microneedle pens could effectively inhibit corneal neovascularization in vivo. Microneedle pens could effectively deliver drugs to thin tissues without impairing merits of using microneedles: localized and minimally invasive delivery.

Acute Central Retinal Artery Occlusion Associated with Livedoid Vasculopathy: A Variant of Sneddon's Syndrome

Livedoid vasculopathy (LV) is characterized by a long history of ulceration of the feet and legs and histopathology indicating a thrombotic process. We report a case of acute central retinal artery occlusion in a 32-year-old woman who had LV. She showed no discernible laboratory abnormalities such as antiphospholipid antibodies and no history of cerebrovascular accidents. Attempted intra-arterial thrombolysis showed no effect in restoring retinal arterial perfusion or vision. The central retinal artery occlusion accompanied by LV in this case could be regarded as a variant form of Sneddons syndrome, which is characterized by livedo reticularis and cerebrovascular accidents.

Optic Disc Edema Responding to Localized Anti-vascular Endothelial Growth Factor Treatment in a Patient with POEMS Syndrome

Dear Editor, Although various underlying conditions such as increased intracranial pressure, inflammation, ischemia, or compression can induce optic disc edema (ODE), all forms of disc swelling ultimately result from blockage of axoplasmic transport [1]. In contrast to these types of ODE, which can generally be managed by systemic treatment of the underlying etiology, we describe a case of ODE treated with intravitreal bevacizumab injection in a patient with polyneuropathy, organomegaly, endocrinopathy, monoclonal gammopathy, and skin changes (POEMS) syndrome, a rare multisystemic disorder associated with a high serum vascular endothelial growth factor (VEGF) level [2]. A 58-year-old man with POEMS syndrome was referred to the ophthalmology department with an 1-month history of bilateral blurred vision and an 1-week history of a round blind spot in the left eye. He also had an 8-year history of POEMS syndrome, which had been in remission for the past 7 years, following treatment with chemotherapy combined with steroids for the first 1 year. He was admitted because of generalized edema and pulmonary hypertension, which began 1 week before. He underwent 3-week systemic treatment with high-dose dexamethasone, which was started one day before the first ophthalmologic examination. He did not have any other past medical problems. Best-corrected visual acuity was 20 / 30 in the right eye and 20 / 50 in the left eye. Fundus examination and optical coherence tomography revealed bilateral ODE and subretinal fluid in the left eye, and fluorescein angiography (FA) demonstrated prominent leakage from the optic disc in both eyes (Fig. 1A-1F). Neither intraretinal microvascular abnormalities nor retinal neovascularization were observed (Fig. 1A and 1F). Fig. 1 (A-F) Initial evaluation with color photos (B,D) and optical coherence tomography (C,E) revealed optic disc edema (ODE) in the right eye (B,C) and in the left eye (D,E). Fluorescein angiography demonstrated prominent leakage in the right eye (A) and in ... After obtaining informed consent, three monthly injections of intravitreal bevacizumab (1.25 mg) were administered to the left eye. Two months after the final injection, both eyes were evaluated with color photos, optical coherence tomography and FA (Fig. 1G-1L). ODE and subretinal fluid showed gradual response and best-corrected visual acuity was improved to 20 / 20 in the left eye (Fig. 1J and 1K). FA also demonstrated decreased disc leakage in the left eye (Fig. 1L). In contrast, right ODE remained unchanged on both optical coherence tomography and FA (Fig. 1G-1I). Eight months after the final injection, ODE and disc leakage were decreased in both eyes and general condition was improved (Fig. 1M-1R). No definite ODE or leakage was observed in the left eye (Fig. 1P-1R). POEMS syndrome is associated with manifestations of increased vascular permeability such as peripheral edema, ascites, pleural effusion, and ODE [3]. Serum VEGF levels of patients with POEMS have been shown to be significantly higher than those of normal controls and other monoclonal gammopathy patients [2]. As VEGF is a potent vascular permeabilizing cytokine, increased serum VEGF in POEMS is thought to play an important role in inducing extravascular volume overload. The responsiveness of ODE to anti-VEGF treatment in our case supports that ODE in POEMS syndrome should also be considered a symptom of extravascular volume overload, similar to peripheral edema, ascites and pleural effusion. It is interesting that ODE and subretinal fluid secondary to optic disc leakage were the only structural ocular abnormalities in POEMS syndrome [3]. No peripheral retinal vessels responded to the increase in serum VEGF. This phenomenon can be explained by the differential actions of VEGF and the anatomy of the optic nerve head (ONH). As VEGF receptors VEGFR1 and VEGFR2 show differential apicobasal distribution, only abluminal VEGF can increase permeability in contrast to luminal VEGF that facilitates cytoprotection instead [4]. Moreover, only the ONH has the border tissue of Elschnig in the prelaminar region, which lacks barrier function and leaks material freely [5]. In POEMS patients, plasma with a high concentration of VEGF would leak into the choroidal interstitial fluid through fenestrated choriocapillaris. VEGF would thus diffuse into the ONH through the border tissue of Elschnig and increases the permeability of capillaries of the ONH by affecting vascular endothelial cells abluminally, while the permeability of the retinal capillaries is not affected by luminal VEGF. The FA findings of our study also showed leakage limited to around the ONH. Our case suggests that intravitreal anti-VEGF injection such as bevacizumab may resolve ODE and lead to visual improvement in POEMS patients.

In Vivo Photoacoustic Imaging of Anterior Ocular Vasculature: A Random Sample Consensus Approach

Visualizing ocular vasculature is important in clinical ophthalmology because ocular circulation abnormalities are early signs of ocular diseases. Photoacoustic microscopy (PAM) images the ocular vasculature without using exogenous contrast agents, avoiding associated side effects. Moreover, 3D PAM images can be useful in understanding vessel-related eye disease. However, the complex structure of the multi-layered vessels still present challenges in evaluating ocular vasculature. In this study, we demonstrate a new method to evaluate blood circulation in the eye by combining in vivo PAM imaging and an ocular surface estimation method based on a machine learning algorithm: a random sample consensus algorithm. By using the developed estimation method, we were able to visualize the PA ocular vascular image intuitively and demonstrate layer-by-layer analysis of injured ocular vasculature. We believe that our method can provide more accurate evaluations of the eye circulation in ophthalmic applications.

Suppression of transient receptor potential canonical channel 4 inhibits vascular endothelial growth factor-induced retinal neovascularization.

Vascular endothelial growth factor (VEGF) is a key regulator of angiogenesis and thus contributes to many vasoproliferative retinopathies including retinopathy of prematurity. Based on the importance of canonical transient receptor potential (TRPC) channels in VEGF signaling, we firstly evaluated the expression of TRPC channels in mouse retina by reverse transcriptase-polymerase chain reaction. All seven TRPC channels were expressed in mouse retina. TRPC4 channels were chosen for further analysis based on their upregulation on hypoxic retina according to the GEO database under the identifier GSE19886. Interestingly, TRPC4 suppression by intravitreal injection of siRNA against mTRPC4 significantly inhibited retinal neovascularization. To further investigate the effect of TRPC4 suppression on neovascularization, human retina microvascular endothelial cells (HRMECs) that are responsible for initiating neovascularization in response to increased VEGF in OIR retina were transfected with siRNA against TRPC4. As we have expected, suppression of TRPC4 effectively inhibited VEGF-induced migration and tube formation as well. Further evaluation on VEGF signaling pathway by western blot analysis of signaling molecules discovered that VEGF-induced activation of ERK, p38 MAPK and AKT signaling pathways were inhibited by suppression of TRPC4. These findings suggest that suppression of TRPC4 could be an alternative therapeutic option for VEGF-induced retinal neovascularization.

Mesenchymal Stromal Cells Inhibit Inflammatory Lymphangiogenesis in the Cornea by Suppressing Macrophage in a TSG-6-Dependent Manner

The cornea is a transparent tissue devoid of blood and lymphatic vessels. However, various inflammatory conditions can cause hemangiogenesis and lymphangiogenesis in the cornea, compromising transparency and visual acuity. Mesenchymal stem/stromal cells (MSCs) have therapeutic potentials in a variety of diseases because of anti-inflammatory properties. Herein, we investigated the effects of MSCs on corneal angiogenesis using a model of suture-induced inflammatory corneal neovascularization. Data demonstrated that an intravenous administration of MSCs suppressed corneal inflammation and neovascularization, inhibiting both hemangiogenesis and lymphangiogenesis. MSCs reduced the levels of vascular endothelial growth factor (VEGF)-C, VEGF-D, Tek, MRC1, and MRC2 in the cornea, which are expressed by pro-angiogenic macrophages. Moreover, the number of CD11b+ monocytes/macrophages in the cornea, spleen, peripheral blood, and draining lymph nodes was decreased by MSCs. Depletion of circulating CD11b+ monocytes by blocking antibodies replicated the effects of MSCs. Importantly, knockdown of tumor necrosis factor alpha (TNF-α)-stimulated gene/protein 6 (TSG-6) in MSCs abrogated the effects of MSCs in inhibiting corneal hemangiogenesis and lymphangiogenesis and monocyte/macrophage infiltration. Together, the results suggest that MSCs inhibit inflammatory neovascularization in the cornea by suppressing pro-angiogenic monocyte/macrophage recruitment in a TSG-6-dependent manner.

Intraocular application of gold nanodisks optically tuned for optical coherence tomography: inhibitory effect on retinal neovascularization without unbearable toxicity

Bare gold nanospheres have been shown to have anti-angiogenic effects but are optically unfavorable because their resonant wavelength lies in the visible spectrum. Here, we design gold nanodisks with a higher scattering capability than gold nanorods and with a resonant wavelength at near-infrared region - the area where the source of light utilized by optical coherence tomography (OCT) lies. With a physical synthesis system, we then fabricate 160-nm-sized gold nanodisks exhibiting resonant wavelength at 830 nm. The synthesized nanoparticles were successfully visualized in in vivo OCT at concentrations as low as 1 pM. After demonstrating their binding ability to vascular endothelial growth factor (VEGF), we show that they suppress VEGF-induced migration of endothelial cells. Finally, we demonstrate that intravitreally injected gold nanodisks attenuate neovascularization of oxygen-induced retinopathy in mice, in a dose dependent manner, such that they are cleared from the vitreous within 2 weeks without histologic or electrophysiologic toxicity.

Disruption of outer blood-retinal barrier by Toxoplasma gondii-infected monocytes is mediated by paracrinely activated FAK signaling.

Ocular toxoplasmosis is mediated by monocytes infected with Toxoplasma gondii that are disseminated to target organs. Although infected monocytes can easily access to outer blood-retinal barrier due to leaky choroidal vasculatures, not much is known about the effect of T. gondii-infected monocytes on outer blood-retinal barrier. We prepared human monocytes, THP-1, infected with T. gondii and human retinal pigment epithelial cells, ARPE-19, grown on transwells as an in vitro model of outer blood-retinal barrier. Exposure to infected monocytes resulted in disruption of tight junction protein, ZO-1, and decrease in transepithelial electrical resistance of retinal pigment epithelium. Supernatants alone separated from infected monocytes also decreased transepithelial electrical resistance and disrupted tight junction protein. Further investigation revealed that the supernatants could activate focal adhesion kinase (FAK) signaling in retinal pigment epithelium and the disruption was attenuated by FAK inhibitor. The disrupted barrier was partly restored by blocking CXCL8, a FAK activating factor secreted by infected monocytes. In this study, we demonstrated that monocytes infected with T. gondii can disrupt outer blood-retinal barrier, which is mediated by paracrinely activated FAK signaling. FAK signaling can be a target of therapeutic approach to prevent negative influence of infected monocytes on outer blood-retinal barrier.

Application of a loop-mediated isothermal amplification (LAMP) assay targeting cox1 gene for the detection of Clonorchis sinensis in human fecal samples

Background Clonorchiasis is prevalent in the Far East, and a major health problem in endemic areas. Infected persons may experience, if not treated, serious complications such as bile stone formation, pyogenic cholangitis, and even cholangiocarcinoma. Early diagnosis and treatment are important to prevent serious complications and, therefore, the simple and reliable diagnostic method is necessary to control clonorchiasis in endemic areas, where resources for the diagnosis are limited. Methodology/Principle findings The loop-mediated isothermal amplification (LAMP) assay has been applied for the detection of Clonorchis sinensis DNA. Six primers targeting eight locations on the cytochrome c oxidase subunit 1 gene of C. sinensis were designed for species-specific amplification using the LAMP assay. The LAMP assay was sensitive enough to detect as little as 100 fg of C. sinensis genomic DNA and the detection limit in 100 mg of stool was as low as one egg. The assay was highly specific because no cross-reactivity was observed with the DNA of other helminths, protozoa or Escherichia coli. Then, LAMP assay was applied to human fecal samples collected from an endemic area of clonorchiasis in Korea. Using samples showing consistent results by both Kato-Katz method and real-time PCR as reference standards, the LAMP assay showed 97.1% (95% CI, 90.1–99.2) of sensitivity and 100% (95% CI, 92.9–100) of specificity. In stool samples with more than 100 eggs per gram of feces, the sensitivity achieved 100%. Conclusions To detect C. sinensis in human fecal samples, the LAMP assay was applied and achieved high sensitivity and specificity. The LAMP assay can be utilized in field laboratories as a powerful tool for diagnosis and epidemiological survey of clonorchiasis.

Melissa officinalis Extract Inhibits Laser-Induced Choroidal Neovascularization in a Rat Model

Purpose This study investigated the effect of Melissa officinalis extract on laser-induced choroidal neovascularization (CNV) in a rat model. The mechanism by which M. officinalis extract acted was also investigated. Methods Experimental CNV was induced by laser photocoagulation in Brown Norway rats. An active fraction of the Melissa leaf extract was orally administered (50 or 100 mg/kg/day) beginning 3 days before laser photocoagulation and ending 14 days after laser photocoagulation. Optical coherence tomography and fluorescein angiography were performed in vivo to evaluate the thickness and leakage of CNV. Choroidal flat mount and histological analysis were conducted to observe the CNV in vitro. Vascular endothelial growth factor (VEGF), matrix metalloproteinase (MMP)-2, and MMP-9 expression were measured in retinal and choroidal-scleral lysates 7 days after laser injury. Moreover, the effect of M. officinalis extract on tertiary-butylhydroperoxide (t-BH)-induced VEGF secretion and mRNA levels of VEGF, MMP-2, and MMP-9 were evaluated in human retinal epithelial cells (ARPE-19) as well as in human umbilical vein endothelial cells (HUVECs). Results The CNV thickness in M. officinalis-treated rats was significantly lower than in vehicle-treated rats by histological analysis. The CNV thickness was 33.93±7.64 µm in the high-dose group (P<0.001), 44.09±12.01 µm in the low-dose group (P = 0.016), and 51.00±12.37 µm in the control group. The proportion of CNV lesions with clinically significant fluorescein leakage was 9.2% in rats treated with high-dose M. officinalis, which was significantly lower than in control rats (53.4%, P<0.001). The levels of VEGF, MMP-2, and MMP-9 were significantly lower in the high-dose group than in the control group. Meanwhile, M. officinalis extract suppressed t-BH-induced transcription of VEGF and MMP-9 in ARPE-19 cells and HUVECs. Conclusions Systemic administration of M. officinalis extract suppressed laser-induced CNV formation in rats. Inhibition of VEGF and MMP-9 via anti-oxidative activity may underlie this effect.