Hideaki Oharazawa

Protection of the Retina by Rapid Diffusion of Hydrogen: Administration of Hydrogen-Loaded Eye Drops in Retinal Ischemia–Reperfusion Injury

PURPOSE Retinal ischemia-reperfusion (I/R) injury by transient elevation of intraocular pressure (IOP) is known to induce neuronal damage through the generation of reactive oxygen species. Study results have indicated that molecular hydrogen (H(2)) is an efficient antioxidant gas that selectively reduces the hydroxyl radical (*OH) and suppresses oxidative stress-induced injury in several organs. This study was conducted to explore the neuroprotective effect of H(2)-loaded eye drops on retinal I/R injury. METHODS Retinal ischemia was induced in rats by raising IOP for 60 minutes. H(2)-loaded eye drops were prepared by dissolving H(2) gas into a saline to saturated level and administered to the ocular surface continuously during the ischemia and/or reperfusion periods. One day after I/R injury, apoptotic cells in the retina were quantified, and oxidative stress was evaluated by markers such as 4-hydroxynonenal and 8-hydroxy-2-deoxyguanosine. Seven days after I/R injury, retinal damage was quantified by measuring the thickness of the retina. RESULTS When H(2)-loaded eye drops were continuously administered, H(2) concentration in the vitreous body immediately increased and I/R-induced *OH level decreased. The drops reduced the number of retinal apoptotic and oxidative stress marker-positive cells and prevented retinal thinning with an accompanying activation of Müller glia, astrocytes, and microglia. The drops improved the recovery of retinal thickness by >70%. CONCLUSIONS H(2) has no known toxic effects on the human body. Thus, the results suggest that H(2)-loaded eye drops are a highly useful neuroprotective and antioxidative therapeutic treatment for acute retinal I/R injury.

Inhibitory Effects of Arg-Gly-Asp (RGD) Peptide on Cell Attachment and Migration in a Human Lens Epithelial Cell Line

Posterior capsule opacification (PCO) after cataract surgery is caused by growth of residual human lens epithelial (HLE) cells on the posterior capsule. We have shown that extracellular matrix (ECM) is an essential factor for HLE cell attachment and migration. The purpose of this study was to examine the inhibitory effects of Arg-Gly-Asp (RGD) peptide on cell attachment and migration in an HLE cell line. HLE cell line cells (SRA 01/04) that were obtained by transfection of large T antigen of SV40 were cultured in the absence of serum. The culture dishes were coated with type IV collagen, laminin or fibronectin, and Gly-Arg-Gly-Asp-Ser-Pro (GRGDSP) RGD peptide (0.1, 0.3, 1.0, 2.0 mg/ml) was added to the medium. The number of attached cells was counted after 90 min of incubation, and the inhibitory effects of GRGDSP RGD peptide on cell attachment were calculated. Cell attachment on the fibronectin-coated dishes was inhibited by GRGDSP RGD peptide at concentrations higher than 0.3 mg/ml; the inhibitory rate was 80% at a concentration of 2.0 mg/ml. The inhibition of cell attachment by GRGDSP RGD peptide on laminin-coated dishes appeared only at a concentration of 2.0 mg/ml, whereas no effects were observed on the type IV collagen-coated dishes. The inhibitory effects of GRGDSP RGD peptide on cell migration were measured in medium containing 2.0 mg/ml of GRGDSP RGD peptide after 1, 3, 5 and 7 days of culture. Cell migration was inhibited by GRGDSP RGD peptide from 1 day of culture on the fibronectin-coated dishes and from 5 days of culture on the laminin-coated dishes, whereas no effects were observed on the type IV collagen-coated dishes. GRGDSP RGD peptide inhibited cell attachment and migration on laminin and fibronectin that have RGD sequences. These data suggested that RGD peptide may have the potential to prevent PCO.

Effect of bottle height on the corneal endothelium during phacoemulsification.

PURPOSE: To directly measure intraocular pressure (IOP) in simulated phacoemulsification and to assess the usefulness of lowering the bottle height in protecting the corneal endothelium in clinical phacoemulsification. SETTING: Nippon Medical School Hospital, Tokyo, Japan. METHODS: Simulated phacoemulsification was performed in porcine eyes with 2 bottle heights, 65.0 cm (BH 65 group) and 19.0 cm (BH 19 group). The IOP was continuously measured with a microprobe. In a clinical study, phacoemulsification was performed with a bottle height of 60.0 cm (BH 60 group) and of 30.0 cm (BH 30 group). One day, 1 week, and 1 and 3 months after surgery, cell density and corneal volumes were measured using specular microscopy and rotating Scheimpflug photography, respectively. RESULTS: In the simulation study, IOP fluctuated between 50 mm Hg and 60 mm Hg in the BH 65 group and between 20 mm Hg and 30 mm Hg in the BH 19 group. In the clinical study of 31 eyes, the rate of cell density decrease was significantly lower in the BH 30 group than in the BH 60 group at all time points. The rate of increase in corneal volume was significantly lower in the BH 30 group than in the BH 60 group at 1 month. CONCLUSIONS: Intraoperative IOP in phacoemulsification with a usual bottle height appeared to exceed the normal range. Phacoemulsification with a low bottle height was less harmful to the corneal endothelium.

Protective effect of molecular hydrogen against oxidative stress caused by peroxynitrite derived from nitric oxide in rat retina.

Oxidative and nitrative processes have an important role in the pathogenesis of glaucomatous neurodegeneration. Oxidative stress occurs when cellular production of reactive oxygen species outweighs the protective capacity of antioxidant defences. Reactive oxygen species are generated as by‐products of cellular metabolism, primarily in the mitochondria. Herein, we present a novel investigation of the effects of molecular hydrogen (H2) on retinal cells exposed to oxidative stress.

Morphological observations of rat corneal endothelial cells after exposure to ozonated solution

PurposeTo determine whether exposure to ozonated solution alters the morphology of corneal endothelial cells in rats and to examine the protective effect of ascorbic acid.MethodsThe anterior chambers of rat eyes were filled with 4 ppm of ozonated solution. Some were left in that state, while others were flushed out either 10, 30, or 60 s after exposure to a balanced salt solution (BSS), or to BSS containing 0.001 M ascorbic acid. Corneal endothelial cells were assessed by scanning and electron microscopy either 1 h or 1 week after treatment, and the expressions of aquaporin (AQ)-1 and zonula occludens (ZO)-1 were determined by immunohistochemistry.ResultsWhen exposure time was longer than 10 s, damaged cell membranes and abnormal organelles were observed 1 h after treatment. The longer the exposure time, the more severe the observed alterations; however, the eyes regained almost their normal state at 1 week. When the BSS contained ascorbic acid, no severe damage was observed under any condition. Normal AQ-1 and ZO-1 expressions were observed even with 60 s of exposure when ascorbic acid was used.ConclusionsA short period of irrigation of the anterior chamber with ozonated solution does not harm the corneal endothelium even when used in combination with ascorbic acid.

Age-related changes of human lens epithelial cells in vivo.

We examined the density and morphology of lens epithelial cells (LECs) in vivo in a group of normal volunteers and cataract patients by using a newly developed noncontact specular microscope. There was a statistically significant decrease in the cell density of LECs in a group of cataract patients over the age of 80 years. The coefficient of variation of the cell area and the number of large black spots that were observed in the enhanced specular images were not related to aging or cataract formation. Our data indicate that the cell density of LECs decreases after reaching the age of 80, but cataract formation does not affect the cell density or the coefficient of variation of the cell area until the age of 80.

Isolated trochlear nerve palsy with perimesencephalic subarachnoid haemorrhage

Perimesencephalic subarachnoid haemorrhage is usually asymptomatic other than meningeal irritation sign. The authors report a case of subarachnoid haemorrhage at the quadrigeminal cistern showing ipsilateral trochlear nerve palsy and discuss the pathogenesis. A 71-year-old man with a history of diabetes mellitus and acute myocardial infarction presented with diplopia. He underwent CT, which revealed subarachnoid haemorrhage at the left quadrigeminal cistern. Neurological examination revealed left isolated trochlear nerve palsy, with results otherwise normal. The diagnosis of perimesencephalic subarachnoid haemorrhage was established on neuroimaging. The amount of haemorrhage is related to symptoms. A dense clot in the quadrigeminal cistern might have been the cause of trochlear nerve palsy.