A Novel Multi-Observation System to Study the Effects of Anterior Ocular Inflammation in Zinn's Zonule Using One Specimen.

Zinn's zonule is a fragile and thin tissue, and little is known about its pathogenesis. The aim of this study was to develop an experimental setup for a comprehensive analysis of Zinn's zonule. Rats were divided into two groups: a control group (n = 4) and an alkali injury group (n = 4). Seven days after injury, the eyes were enucleated, the anterior eye was dissected and embedded in gelatin, and macroscopic observations were made. The gelatin specimens were then embedded in paraffin and observed in detail by low-vacuum scanning electron microscopy, immunofluorescence, and quantitative reverse transcription polymerase chain reaction (RT-qPCR). The results show qualitative changes in Zinn's zonules in both macroscopic and microscopic observations. In addition, macrophage infiltration and increased matrix metalloproteinase 2 (MMP2) expression were observed in the injured group, consistent with the RT-qPCR results. The experimental system in this study allowed us to capture the morphological and molecular biological changes of Zinn's zonule and to gain insight into its pathogenesis. In conclusion, this study presents a new experimental setup for the comprehensive analysis of the rat Zinn's zonule. The results suggest that this system can be used in the future to study and analyze a variety of paraffin-embedded tissues and specimens.

Disulfiram Ophthalmic Solution Inhibited Macrophage Infiltration by Suppressing Macrophage Pseudopodia Formation in a Rat Corneal Alkali Burn Model.

FROUNT is an intracellular protein that promotes pseudopodia formation by binding to the chemokine receptors CCR2 and CCR5 on macrophages. Recently, disulfiram (DSF), a drug treatment for alcoholism, was found to have FROUNT inhibitory activity. In this study, we investigated the effect of DSF eye drops in a rat corneal alkali burn model. After alkali burn, 0.5% DSF eye drops (DSF group) and vehicle eye drops (Vehicle group) were administered twice daily. Immunohistochemical observations and real-time reverse transcription-polymerase chain reaction (RT-PCR) analyses were performed at 6 h and 1, 4, and 7 days after alkali burn. Results showed a significant decrease in macrophage accumulation in the cornea in the DSF group, but no difference in neutrophils. RT-PCR showed decreased expression of macrophage-associated cytokines in the DSF group. Corneal scarring and neovascularization were also suppressed in the DSF group. Low-vacuum scanning electron microscopy imaging showed that macrophage length was significantly shorter in the DSF group, reflecting the reduced extension of pseudopodia. These results suggest that DSF inhibited macrophage infiltration by suppressing macrophage pseudopodia formation.

Prophylactic Instillation of Hydrogen-Rich Water Decreases Corneal Inflammation and Promotes Wound Healing by Activating Antioxidant Activity in a Rat Alkali Burn Model.

Many studies have demonstrated the therapeutic effects of hydrogen in pathological conditions such as inflammation; however, little is known about its prophylactic effects. The purpose of this study is to investigate the prophylactic effects of hydrogen-rich water instillation in a rat corneal alkali burn model. Hydrogen-rich water (hydrogen group) or physiological saline (vehicle group) was instilled continuously to the normal rat cornea for 5 min. At 6 h after instillation, the cornea was exposed to alkali. The area of corneal epithelial defect (CED) was measured every 6 h until 24 h after alkali exposure. In addition, at 6 and 24 h after injury, histological and immunohistochemical observations were made and real-time reverse transcription polymerase chain reaction (RT-PCR) was performed to investigate superoxide dismutase enzyme (SOD)1, SOD2, and peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α) mRNA expression. CED at 12 h and the number of inflammatory infiltrating cells at 6 h after injury were significantly smaller in the hydrogen group than the vehicle group. Furthermore, SOD1 expression was significantly higher in the hydrogen group than the vehicle group at both 6 and 24 h, and the number of PGC-1α-positive cells was significantly larger in the hydrogen group than the vehicle group at 6 h after injury. In this model, prophylactic instillation of hydrogen-rich water suppressed alkali burn-induced inflammation, likely by upregulating expression of antioxidants such as SOD1 and PGC-1α. Hydrogen has not only therapeutic potential but also prophylactic effects that may suppress corneal scarring following injury and promote wound healing.

Dietary GABA induces endogenous synthesis of a novel imidazole peptide homocarnosine in mouse skeletal muscles.

Carnosine (ß-alanyl-L-histidine) is an imidazole dipeptide present at high concentrations in skeletal muscles, where it plays a beneficial role. However, oral intake of carnosine or ß-alanine to increase skeletal muscle carnosine levels has disadvantages such as low efficiency and side effects. Therefore, we proposed homocarnosine (γ-aminobutyryl-L-histidine) as a novel alternative imidazole peptide for skeletal muscle based on its structural similarity to carnosine. To induce endogenous homocarnosine synthesis in skeletal muscles, mice were fed a basal diet mixed with 0, 0.5, 2, or 5% γ-aminobutyric acid (GABA) for 6 weeks. As expected, in the control group (0% GABA), GABA and homocarnosine were present in trace concentrations. Skeletal muscle homocarnosine levels were significantly increased in the 2% and 5% GABA intake groups (tenfold, P < 0.01 and 53-fold, P < 0.01; respectively) relative to those of the control group, whereas 0.5% GABA intake induced no such effect. GABA intake had no effect on the levels of carnosine, anserine, and ß-alanine. Vigabatrin (inhibitor of GABA transaminase (GABA-T)) administration to mice receiving 2% GABA intake for 2 weeks led to GABA-T inhibition in the liver. Subsequently, a 43-fold increase in circulating GABA levels and a tendency increase in skeletal muscle homocarnosine levels were observed. Therefore, skeletal muscle homocarnosine synthesis can be induced by supplying its substrate GABA in tissues. As GABA availability is tightly regulated by GABA-T via GABA degradation, inhibitors of GABA or ß-alanine degradation could be novel potential interventions for increasing skeletal muscle imidazole dipeptides.

Combination of Peroxisome Proliferator-Activated Receptor (PPAR) Alpha and Gamma Agonists Prevents Corneal Inflammation and Neovascularization in a Rat Alkali Burn Model.

Peroxisome proliferator-activated receptor alpha (PPARα) and gamma (PPARγ) agonists have anti-inflammatory and anti-neovascularization effects, but few reports have tested the combination of PPARα and PPARγ agonists. In this study, we investigated the therapeutic effects of ophthalmic solutions of agonists of PPARα, PPARγ, and the combination in a rat corneal alkali burn model. After alkali injury, an ophthalmic solution of 0.05% fenofibrate (PPARα group), 0.1% pioglitazone (PPARγ group), 0.05% fenofibrate + 0.1% pioglitazone (PPARα+γ group), or vehicle (vehicle group) was topically instilled onto the rat's cornea twice a day. After instillation, upregulation was seen of PPAR mRNA corresponding to each agonist group. Administration of agonists for PPARα, PPARγ, and PPARα+γ suppressed inflammatory cells, neovascularization, and fibrotic changes. In addition, the PPARγ agonist upregulated M2 macrophages, which contributed to wound healing, whereas the PPARα agonist suppressed immature blood vessels in the early phase. Administration of PPARα+γ agonists showed therapeutic effects in corneal wound healing, combining the characteristics of both PPARα and PPARγ agonists. The results indicate that the combination of PPARα and γ agonists may be a new therapeutic strategy.

Peroxisome Proliferator-Activated Receptor Beta/Delta Agonist Suppresses Inflammation and Promotes Neovascularization.

The effects of peroxisome proliferator-activated receptor (PPAR)ß/δ ophthalmic solution were investigated in a rat corneal alkali burn model. After alkali injury, GW501516 (PPARß/δ agonist) or vehicle ophthalmic solution was topically instilled onto the rat's cornea twice a day until day 7. Pathological findings were evaluated, and real-time reverse transcription polymerase chain reaction was performed. GW501516 strongly suppressed infiltration of neutrophils and pan-macrophages, and reduced the mRNA expression of interleukin-6, interleukin-1ß, tumor necrosis factor alpha, and nuclear factor-kappa B. On the other hand, GW501516 promoted infiltration of M2 macrophages, infiltration of vascular endothelial cells associated with neovascularization in the wounded area, and expression of vascular endothelial growth factor A mRNA. However, 7-day administration of GW501516 did not promote neovascularization in uninjured normal corneas. Thus, the PPARß/δ ligand suppressed inflammation and promoted neovascularization in the corneal wound healing process. These results will help to elucidate the role of PPARß/δ in the field of ophthalmology.

Transgenic mice specifically expressing amphiregulin in white adipose tissue showed less adipose tissue mass.

To determine adipocytokines that play a regulatory role during obesity development, we explored the genes that encode growth factors and investigated the physiological functions for adipose tissue development. Here, we isolated amphiregulin (Areg) gene whose expression was significantly up-regulated in obese adipose tissues. Areg mRNA level was positively correlated with macrophage marker gene expression in adipose tissues in vivo. Unexpectedly, Areg transgenic mice showed less adipose tissue mass with increased mRNA expression levels of Tnf-α and peroxisome proliferator-activated receptor γ coactivator 1α (Pgc-1α) and delayed white adipose tissue development during the convalescent stage in a dextran sodium sulfate-induced colitis model. This study showed that Areg mRNA expression was significantly up-regulated in obese adipose tissues and over-expression of Areg in white adipose tissue caused less adipose tissue mass.

PPARα Agonist Suppresses Inflammation after Corneal Alkali Burn by Suppressing Proinflammatory Cytokines, MCP-1, and Nuclear Translocation of NF-κB.

We investigated the effect of a peroxisome proliferator-activated receptor α (PPARα) agonist after corneal alkali injury. Fenofibrate 0.05% (PPARα agonist group) or vehicle (Vehicle group) was topically instilled onto the rat cornea after injury. Histological, immunohistochemical, and real-time reverse transcription PCR analyses were performed. PPARα-positive cells were observed among basal cells of the corneal epithelium in normal and alkali-burned corneas. The number of infiltrating neutrophils and macrophages at the corneal limbus was lower in the PPARα agonist group. Interleukin-1ß (IL-1ß), IL-6, IL-8, monocyte chemoattractant protein-1 (MCP-1), and vascular endothelial growth factor-An mRNA expression was suppressed in the PPARα agonist group compared to the Vehicle group. mRNA levels of nuclear factor kappa B (NF-κB) in corneal tissue were not different. However, NF-κB was expressed in the cytoplasm of basal cells in the PPARα agonist group and in the nucleus in the Vehicle group. MCP-1 was more weakly expressed in the PPARα agonist group. The PPARα agonist inhibited inflammation during the early phase after injury. Anti-inflammatory effects of the PPARα agonist included prevention of up-regulation of proinflammatory cytokines and MCP-1, and prevention of inflammatory cell infiltration into the injured cornea. Thus, a PPARα agonist may be a promising treatment for corneal injury.

Trimebutine prevents corneal inflammation in a rat alkali burn model.

Alkaline burns to the cornea lead to loss of corneal transparency, which is essential for normal vision. We used a rat corneal alkaline burn model to investigate the effect of ophthalmic trimebutine solution on healing wounds caused by alkaline burns. Trimebutine, an inhibitor of the high-mobility group box 1-receptor for advanced glycation end products, when topically applied to the burned cornea, suppressed macrophage infiltration in the early phase and neutrophil infiltration in the late phase at the wound site. It also inhibited neovascularization and myofibroblast development in the late phase. Furthermore, trimebutine effectively inhibited interleukin-1ß expression in the injured cornea. It reduced scar formation by decreasing the expression of type III collagen. These findings suggest that trimebutine may represent a novel therapeutic strategy for corneal wounds, not only through its anti-inflammatory effects but also by preventing neovascularization.

A novel initial wiring technique for chronic total occlusion of the superficial femoral artery using the structural features of a polymer jacket guidewire.

BACKGROUND: To evaluate the efficacy of the GLadIus MG drilLINg technique (GLIMGLIN), a novel initial wiring technique using the Gladius MG™ structural features, for crossing the superficial femoral artery (SFA) with chronic total occlusion (CTO). METHODS: This retrospective, single-center study enrolled 27 symptomatic patients (mean age 77.4 ± 8.5 years; 20 men) with de novo SFA CTO (mean CTO length 16.1 ± 8.9 cm) who underwent GLIMGLIN as the initial wiring between January 2020 and December 2021. The success of GLIMGLIN was defined when the wire crossing was completed using a Gladius MG™ and a microcatheter without any additional devices and techniques. RESULTS: The success rate of GLIMGLIN was 48.1%. Intravascular ultrasound findings showed complete true lumen passage in the GLIMGLIN success group. Compared to the failure group, the proximal (6.3 ± 0.8 vs. 5.5 ± 0.9 mm, p = 0.02) and distal (5.9 ± 0.5 vs. 5.4 ± 0.6 mm, p = 0.02) reference vessel diameters were significantly larger, and the rate of calcium angle > 180° was significantly lower (30.8 vs. 71.4%, p = 0.04) in the success group. No significant difference was shown in the CTO length between two groups. Total wiring time, total procedure time, and fluoroscopic time were significantly shorter in the success group. CONCLUSIONS: GLIMGLIN may enable operators to perform CTO wiring easily and efficiently in selected cases.

Statistical Analysis of Factors Affecting Surgically Induced Astigmatism Following Trabeculectomy.

Purpose: To objectively evaluate surgically induced astigmatism (SIA) after trabeculectomy with mitomycin C and investigate the relationships between SIA and various factors. Patients and Methods: This retrospective study included the right eyes of 66 consecutive patients who underwent standard trabeculectomy performed in the superior temporal quadrant for the first time by a single surgeon. Keratometry recordings made before surgery and 3 months after surgery were collected to calculate the SIA in each patient. The arithmetic mean of SIA (M-SIA) and the centroid of SIA (C-SIA) were determined using vector analysis. The relationships between the magnitude of SIA and the following possible related factors were assessed: age, sex, pre-operative corneal astigmatism, pre-operative intraocular pressure (IOP), 3-month postoperative IOP, pre-operative best-corrected visual acuity (BCVA), 3-month postoperative BCVA, the number of total scleral flap sutures (T-SFS), the number of leftover scleral flap sutures without laser suture lysis at 3 months postoperatively (L-SFS), shape of the scleral flap (triangle or trapezoid), and incision type of the conjunctival flap (fornix- or limbal-based). Results: The mean (± standard deviation) M-SIA was 1.00 ± 0.85 D, and the mean C-SIA was 0.34 ± 1.28 D at 104°. The direction of C-SIA showed a trend of corneal steepening to the superior temporal location, in the direction of the scleral flap location. There were significant correlations of the magnitudes of SIA with the number of T-SFS (P = 0.001) and the number of L-SFS (P < 0.001). Conclusion: Trabeculectomy induced SIA in the direction of the scleral flap location, and scleral sutures are significantly associated with the SIA. The scleral suture may play a key role in steepening the cornea toward the scleral flap direction in post-trabeculectomy patients.

A clinical trial for therapeutic drug monitoring using microchip-based fluorescence polarization immunoassay.

Microchip analysis is a promising method for therapeutic drug monitoring. This led us to evaluate a microchip-based fluorescence polarization immunoassay (FPIA) system for point-of-care testing on patients being treated with theophylline. The sera were collected from 20 patients being treated with theophylline. Fluorescence polarization was measured on the microchip and theophylline concentrations in serum were obtained. Regression analysis of the correlations was done between the results given by the microchip-based FPIA and the conventional cloned enzyme donor immunoassay (CEDIA), and between the results given by the microchip-based FPIA and the conventional particle-enhanced turbidimetric inhibition immunoassay (PETINIA). We successfully carried out a quantitative analysis of theophylline in serum at values near its therapeutic range in 65 s. The results obtained by the microchip-based FPIA correlated well with CEDIA and PETINIA results; the correlation coefficients (R(2)) were 0.986 and 0.989, respectively. The FPIA system is a simple and rapid method for point-of-care testing of drugs in serum, and its accuracy is the same as the conventional CEDIA and PETINIA. It is essential to use real samples from patients and to confirm good correlations with conventional methods for a study on the realization of microchip.

Effects of Selective Peroxisome Proliferator Activated Receptor Agonists on Corneal Epithelial Wound Healing.

The effects of each subtype-selective peroxisome proliferator activated receptor (PPAR) agonist (α, ß/δ, γ) on corneal epithelial wound healing were investigated using a rat corneal alkali burn model. After the alkali burn, each PPAR agonist or vehicle ophthalmic solution was instilled topically onto the rat's cornea. Corneal epithelial healing processes were evaluated by fluorescein staining. Pathological analyses and real-time reverse transcription polymerase chain reactions were performed to evaluate Ki67 (proliferative maker) expression and inflammatory findings. The area of the corneal epithelial defect at 12 h and 24 h after the alkali burn was significantly smaller in each PPAR group than in the vehicle group. Ki67 mRNA expression was increased in the PPARß/δ group, whereas mRNA expressions of inflammatory cytokines were suppressed in all of the PPAR agonist groups. Nuclear factor kappa B (NF-κB) was the most suppressed in the PPARγ group. The accelerated corneal epithelial healing effects of each PPAR ligand were thought to be related to the promotion of proliferative capacity and inhibition of inflammation.

Changes in the Ganglion Cell Complex after Inner Limiting Membrane Peeling for Epiretinal Membrane in Glaucoma Patients.

BACKGROUND: Epiretinal membrane (ERM) is a disease that affects the vitreoretinal interface and causes metamorphopsia, anorthopia, and decreased visual acuity. In this study, ERM patients who underwent internal limiting membrane (ILM) peeling were classified as those with glaucoma (Group G) and a control group (Group C). Changes in ganglion cell complex (GCC) thickness were compared between these groups to investigate whether such changes had an effect on progression of glaucoma from structural change. METHODS: This was a retrospective, observational study that included 27 eyes of 27 patients. Group C included 22 eyes, and Group G included 5 eyes. Patients underwent ILM peeling, and cataract surgery was combined with vitrectomy for 16 phakic eyes; 2 phakic eyes and 9 aphakic eyes were treated only with vitrectomy. GCC thickness was measured preoperatively and at 2 weeks and 1, 3, and 6 months postoperatively, and these values and the rates of thinning were compared between the two groups. RESULTS: The mean age of patients was 66.7±12.8 years (range 30-84 years). There was no significant difference between groups in the thickness of the GCC or its rate of thinning after ILM peeling. CONCLUSIONS: The present results suggest that this procedure does not cause structural exacerbation of glaucoma in glaucoma patients. Although further studies of the functional effects of ILM peeling are required, the present results suggest that there is no significant difference between the two groups.

Optical Coherence Tomography Angiography of Nonarteritic Cilioretinal Artery Occlusion Alone.

Cilioretinal artery occlusion (CLRAO) is a rare disease. Here, we report the case of a 70-year-old man with nonarteritic cilioretinal artery occlusion alone. The patient was allergic to fluorescein. Therefore, we followed the retinal circulation with optical coherence tomography angiography (OCTA). OCTA at 40 days postonset showed partial improvement in the retinal circulation.

Changes in Tear Osmolarity after Cataract Surgery.

BACKGROUND: The purpose of this study was to examine changes in the ocular surface before and after phacoemulsification with small incisions and to examine the changes in tear osmolarity. METHODS: This was a prospective, observational study involving 55 eyes of 39 patients (19 male, 20 female patients; average age 72.0±7.3 years) who had cataract surgery at a Nippon Medical School Hospital between December 2013 and June 2018. Compromised tear dynamics were determined by the Schirmer test or the tear break-up time (BUT). An abnormal ocular surface was identified by positive vital staining with fluorescein or lissamine green. Moreover, tear osmolarity (Tosm) and corneal sensitivity were measured. All assessments were done preoperatively and 1 and 4 weeks (P1W and P4W) after the surgery. RESULTS: None of the operations had any complications. Operating time was 17.8±9.3 minutes. BUT was significantly decreased at P1W, and it recovered at P4W. The Schirmer test did not change significantly. The fluorescein staining score (FSS) increased significantly at P1W and recovered at P4W. The Lissamine green score (LSS) did not change significantly. Tear osmolarity increased significantly at P1W and did not recover at P4W. Corneal sensitivity decreased significantly at P1W and recovered at P4W. CONCLUSION: In the present study, there were temporary changes in dry eye-related examinations including tear osmolarity after cataract surgery. In particular, tear osmolarity increased significantly 4 weeks after surgery compared to before surgery, and it showed long-term changes, unlike other factors. After cataract surgery, tear osmolarity, BUT, and FSS increase, resulting in dry eye symptoms. Therefore, it is necessary to pay attention to discomfortable eye symptoms of patients after cataract surgery.

Brain-derived Neurotrophic Factor in the Aqueous Humor of Glaucoma Patients.

BACKGROUND: Brain-derived neurotrophic factor (BDNF) may be involved in the pathogenesis of glaucoma. BDNF concentrations reported in previous studies have varied widely, and the concentration of BDNF in aqueous humor is unknown. In this study, BDNF concentrations in the aqueous humor of glaucoma patients and control patients were measured with ELISA kits. METHODS: This prospective, observational study examined BDNF levels in aqueous humor in 62 eyes of 43 patients who underwent cataract surgery or trabeculectomy (11 glaucoma patients and 32 non-glaucoma cataract patients as controls). BDNF concentrations were examined by 4 different enzyme-linked immunosorbent assay (ELISA) techniques. RESULTS: The mean ± SD patient age was 72.0 ± 10.1 (range 35 to 87) years. Two of the techniques detected no BDNF in aqueous humor in any samples (n=3 and n=9, respectively); the average value was less than zero. An ultrasensitive ELISA kit did not yield reliable measurements. Finally, in an even more sensitive ELISA (Simoa-HD1), performed by an outside contractor, 25 (54.3%) eyes were below the detection limit, including 20 (55.6%) control and 5 (50%) glaucoma cases. For eyes with detectable BDNF, the overall BDNF concentration was 0.158 pg/mL (n=21): 0.196 pg/mL (n=16) in controls and 0.034 pg/mL (n=5) in glaucoma cases. CONCLUSIONS: BDNF level in aqueous humor varies widely.

Observation of Corneal Wound Healing and Angiogenesis Using Low-Vacuum Scanning Electron Microscopy.

Purpose: Wound healing processes in a rat corneal alkali burn model were observed using low-vacuum scanning electron microscopy (LV-SEM), a new observation method that can use paraffin sections for light microscopic immunostaining. Methods: Injured cornea was observed under immunohistochemistry, LV-SEM, and transmission electron microscopy. In LV-SEM, periodic acid-methenamine silver staining was used to observe collagen and platinum blue staining was used to observe vascular endothelial cells. Analyses of the messenger RNA expression involved in neovascularization processes after wound creation were also performed. Results: LV-SEM depicted progression of corneal wound healing in a stereoscopic fashion. In neovascularization processes after wound creation, LV-SEM with osmification clearly demonstrated detachment of pericytes from the vascular endothelial cells, in association with up-regulation of angiopoietin-2 messenger RNA expression. Conclusions: LV-SEM enables high magnification observation of paraffin sections used for immunohistochemistry. LV-SEM provides easy, detailed observations and offers a promising new observational modality in the field of ophthalmology. Translational Relevance: High magnification analysis was easily available using LV-SEM with conventional paraffin sections for light microscopy.

Hydrogen promotes the activation of Cu, Zn superoxide dismutase in a rat corneal alkali-burn model.

AIM: To investigate the effects of hydrogen (H2) on Cu, Zn superoxide dismutase (SOD1) activation in a rat model of corneal alkali burn. METHODS: In each rat, one cornea was subjected to alkali exposure. Physiological saline (saline group) or H2-dissolved saline (H2 group) was instilled continuously on the cornea for 5min before and after alkali exposure. Inflammatory cells, neovascularization, and cytoplasmic SOD1 levels were evaluated immunohistochemically in enucleated eyes from both groups. Three-dimensional ultrastructural tissue changes in the eyes were analyzed using low-vacuum scanning electron microscopy. RESULTS: The numbers of both inflammatory and vascular endothelial cells were significantly reduced in the corneas of the H2 group (P<0.01). Furthermore, H2 treatment increased both cytoplasmic SOD1 levels (P<0.01) and activity in corneal epithelial cells (P<0.01). Notably, the SOD1 activity level in the H2 group was approximately 2.5-fold greater than that in the saline group. CONCLUSION: H2 treatment suppresses inflammation and neovascularization in the injured cornea and indirectly suppresses oxidative insult to the cornea by upregulating the SOD1 enzyme protein level and activity.

Effects of Hydrogen in Prevention of Corneal Endothelial Damage During Phacoemulsification: A Prospective Randomized Clinical Trial.

PURPOSE: Hydrogen (H2) has been reported to scavenge free radicals, particularly the hydroxyl radical (·OH). Ultrasound oscillation in an aqueous solution produces ·OH. Our recent study demonstrated that H2 dissolved in an irrigation solution prevented corneal endothelial damage during phacoemulsification in an animal model. We examined the effects of H2 during clinical phacoemulsification. DESIGN: A single-center, prospective, randomized, double-masked clinical trial. METHODS: Thirty-two patients who had cataracts of similar nucleus hardness in both eyes (age: 75.4±7.68 years; 17 males, 15 females) were recruited. Phacoemulsification was performed using a solution of dissolved H2 in one eye, and a conventional solution in the contralateral eye. Endothelial cell density (ECD) at the center of the cornea was measured using noncontact specular microscopy preoperatively and at 1 day, 1 week, and 3 weeks postoperatively. RESULTS: Reduction rates of ECD (mean ± standard deviation) were 16.0%±15.7% at 1 day, 15.4%±16.1% at 1 week, and 18.4%±14.9% at 3 weeks in the control group, compared to 6.5%±8.7% at 1 day (P = .003), 9.3%±11.0% at 1 week (P = .039), and 8.5%±10.5% at 3 weeks (P = .004) in the H2 groups. These rates were significantly smaller in the H2 group at all time points. CONCLUSIONS: H2 dissolved in irrigation solution reduced corneal endothelial damage during phacoemulsification. This suggests that a considerable part of the corneal endothelial damage during phacoemulsification is caused by oxidative stress, and that H2 is useful in clinical phacoemulsification.