January 2010
Volume 51, Issue 1
Free
Retina  |   January 2010
Protection of the Retina by Rapid Diffusion of Hydrogen: Administration of Hydrogen-Loaded Eye Drops in Retinal Ischemia–Reperfusion Injury
Author Affiliations & Notes
  • Hideaki Oharazawa
    From the Department of Ophthalmology, Musashikosugi Hospital, Nippon Medical School, Kanagawa, Japan;
  • Tsutomu Igarashi
    the Department of Ophthalmology, Nippon Medical School, Tokyo, Japan; and
  • Takashi Yokota
    the Department of Molecular Biology,
  • Hiroaki Fujii
    From the Department of Ophthalmology, Musashikosugi Hospital, Nippon Medical School, Kanagawa, Japan;
  • Hisaharu Suzuki
    the Department of Ophthalmology, Nippon Medical School, Tokyo, Japan; and
  • Mitsuru Machide
    The Center of Molecular Hydrogen Medicine, and
  • Hiroshi Takahashi
    the Department of Ophthalmology, Nippon Medical School, Tokyo, Japan; and
  • Shigeo Ohta
    the Department of Biochemistry and Cell Biology, Institute of Development and Aging Sciences, Nippon Medical School, Kanagawa, Japan.
  • Ikuroh Ohsawa
    The Center of Molecular Hydrogen Medicine, and
  • Corresponding author: Ikuroh Ohsawa, The Center of Molecular Hydrogen Medicine, Institute of Development and Aging Sciences, Nippon Medical School, 1-396 Kosugi-cho, Nakahara-ku, Kawasaki, 211-8533 Japan; iohsawa@nms.ac.jp
Investigative Ophthalmology & Visual Science January 2010, Vol.51, 487-492. doi:10.1167/iovs.09-4089
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      Hideaki Oharazawa, Tsutomu Igarashi, Takashi Yokota, Hiroaki Fujii, Hisaharu Suzuki, Mitsuru Machide, Hiroshi Takahashi, Shigeo Ohta, Ikuroh Ohsawa; Protection of the Retina by Rapid Diffusion of Hydrogen: Administration of Hydrogen-Loaded Eye Drops in Retinal Ischemia–Reperfusion Injury. Invest. Ophthalmol. Vis. Sci. 2010;51(1):487-492. doi: 10.1167/iovs.09-4089.

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      © 2016 Association for Research in Vision and Ophthalmology.

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Abstract

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 (H2) 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 H2-loaded eye drops on retinal I/R injury.

Methods.: Retinal ischemia was induced in rats by raising IOP for 60 minutes. H2-loaded eye drops were prepared by dissolving H2 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 H2-loaded eye drops were continuously administered, H2 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.: H2 has no known toxic effects on the human body. Thus, the results suggest that H2-loaded eye drops are a highly useful neuroprotective and antioxidative therapeutic treatment for acute retinal I/R injury.

Retinal ischemia-reperfusion (I/R) injury by transient elevation of intraocular pressure (IOP) in animal models is known to induce necrosis and apoptosis of cells and significant reductions in thickness in multiple layers of the retina. 1,2 Clinically, these features closely resemble several diseases such as acute angle-closure glaucoma, retinal artery occlusion, and amaurosis fugax. 3 It can irreversibly damage the retina, causing visual impairment and blindness. Immediate mechanisms of I/R injury involve the formation of reactive oxygen species (ROS), 4 which has been considered to contribute to the pathogenesis of many neurodegenerative diseases, including glaucomatous neurodegeneration. 5 Endogenous antioxidant enzymes and organic free radical scavengers can retard or prevent neuronal damages of retinal I/R injury in many animal models. 613 One highly reactive ROS, hydroxyl radical (·OH), is generated during the early phase of reperfusion after ischemia and a major cause of retinal injury. 1416 ·OH attacks lipids, proteins and nucleic acids causing irreversible cellular damage. 
In the past two decades, much attention has been focused on the use of several pharmaceutical gaseous molecules to attenuate oxidative stress. 17 A variety of gas delivery systems are used and under development for safe and effective administration of medical gases. We have reported that H2 selectively reduces ·OH and peroxynitrite without affecting other oxygen-derived free radicals. 18 Inhalation of H2 gas has been demonstrated to limit the infarct volume of the brain, heart, and liver by reducing I/R injury 1821 and can ameliorate intestinal transplant injury. 22 Moreover, the consumption of water with dissolved H2 to a saturated level prevents stress-induced cognitive decline and 6-hydroxydopamine-induced nigrostriatal degeneration. 23,24 One clinical trial demonstrated a decrease in low-density lipoprotein after drinking H2-loaded water. 25 H2 has the potential to easily diffuse into organs and no known toxic effects on the human body. 18  
We have therefore developed a simple and effective method to deliver H2 into lesions. The method is H2-loaded eye drops, which are convenient, compared with the inhalation of H2 gas, for the treatment of eye diseases. In this article, we demonstrate that the continuous administration of H2-loaded eye drops immediately increases H2 concentration in the vitreous body and prevents I/R-induced oxidative stress, leading to a decrease in apoptotic cell death in the retina and a decrease in retinal thinning with glial responses. 
Methods
Administration of H2 and Measurement of Its Concentration
H2-loaded eye drops were prepared by bubbling H2 gas (flow rate: 1 L/min) through 400 mL of normal saline solution with stirring for 10 minutes to a saturated level (Fig. 1A), and then stored in an aluminum foil bag (Fig. 1B; Hosokawa Yoko, Tokyo, Japan) with no dead volume. The concentration of H2 in the bag slowly decreased with a half-life of approximately 3 months. Freshly prepared H2-loaded eye drops were administered to the ocular surface continuously (4 mL/min) with a dropper connected to the aluminum foil bag during the ischemia and/or reperfusion periods. The H2 dissolved in saline solution was measured by using a needle-type H2 sensor (Unisense, Aarhus N, Denmark). The H2 concentration on the ocular surface was measured by touching the sensor to the surface. H2 concentration was measured in the vitreous body by inserting the sensor into the vitreous body through the sclera. 
To investigate the effect of H2-loaded eye drops on retinal I/R injury, we applied them using four different time courses (see Fig. 4A): duration F, eye drops with and without H2 were applied during an entire 90-minute process (60 minutes of ischemia followed by 30 minutes of reperfusion); duration I, eye drops with H2 were applied only during ischemia; duration R, eye drops with H2 were applied only after reperfusion; and duration I/R, eye drops with H2 were applied for 10 minutes before and 30 minutes after reperfusion. 
Induction of I/R Injury
Retinal I/R injury was induced essentially as described previously. 2,26 Seven-week-old male Sprague–Dawley rats weighing 200 to 250 g were anesthetized with an intraperitoneal injection of pentobarbital (100 mg/kg), and the pupils were dilated with topical phenylephrine hydrochloride and tropicamide. After topical application of 0.4% oxybuprocaine hydrochloride, the anterior chamber was cannulated with a 30-gauge infusion needle connected to a normal saline reservoir. The IOP was raised to 110 mm Hg for 60 minutes by elevating the saline reservoir. Body temperature was maintained at 37.0 ± 0.5°C with a rectal thermometer probe and a heating pad during the experimental period. Retinal ischemia was confirmed by whitening of the iris and fundus. After 60 minutes of ischemia, the needle was withdrawn from the anterior chamber and the intraocular pressure was normalized. The animals were euthanatized with an overdose of anesthesia after reperfusion, and the eyes were immediately enucleated. All animals were treated in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. The studies were approved by the Animal Care and Use Committee of Nippon Medical School. All experiments were performed by examiners blinded to the genotypes or treatments of the rat. 
Detection of ·OH
The procedure for the measurement of accumulated ·OH in the eye is similar to that previously described with modifications. 27 We used 2-[6-(4′-hydroxy)phenoxy-3H-xanthen-3-on-9-yl] benzoate (HPF; Daiichi Pure Chemicals, Tokyo, Japan), which detects highly reactive ROS including ·OH, as a fluorescence probe. 28 HPF (4 μL, 1 mM) was given intravitreally just before the induction of ischemia. Rats were killed after 60 minutes of ischemia followed by 15 minutes of reperfusion. Retinas were quickly removed and flat mounted without fixation. The fluorescence images were acquired by using a laser scanning confocal microscope. The acquired images were analyzed by quantitative comparisons of the relative fluorescence intensity of retinas between groups (NIH Image software, developed by Wayne Rasband, National Institutes of Health, Bethesda, MD; available at http://rsb.info.nih.gov/ij/index.html). 
Histopathologic and Morphometric Study
Eyes were enucleated 7 days after reperfusion and fixed in 1% glutaraldehyde and 4% paraformaldehyde (PFA) in 0.1 M phosphate-buffered saline (PBS) for 30 minutes, and the anterior segments were removed. Corneas and lenses were discarded. The entire eye cups were further fixed in the same solution overnight and then transferred to 30% sucrose for cryoprotection. Cryosections (10 μm thick) were cut along the vertical meridian of the eye, passing through the optic nerve head, and were stained with hematoxylin and eosin (H&E). Retinal damage was assessed by measuring the thickness of the retina. 1 The thickness is defined as the total width between the inner limiting membrane to the interface of the outer plexiform layer and the outer nuclear layer. These measurements were made at a distance within 1 to 2 mm from the optic disc using a light microscope. The value was averaged from four measurements in the temporal and nasal hemispheres of three different sections. 
TUNEL Assay and Immunohistochemical Staining
One day or 7 days after reperfusion, the eyes were immediately enucleated. For TUNEL assay and the staining of reactive gliosis markers, they were fixed in 4% PFA, and for staining of oxidative stress markers they were fixed in Bouin's fluid for 30 minutes. Next, the anterior segments were removed and the corneas and lenses were discarded. For the TUNEL assay and for the staining of reactive gliosis markers, the obtained entire eye cups were further fixed in the same solution overnight. For the staining of oxidative stress markers, they were further fixed in the same solution for 2 hours. After cryoprotection with 30% sucrose, cryosections (10 μm thick) were cut along the vertical meridian of the eye, passing through the optic nerve head. TUNEL staining was performed with an apoptosis detection kit according to the supplier's instructions (Chemicon, Norcross, GA). 29 The numbers of TUNEL-positive cells in the retina were counted at a final magnification ×200 for each section using a light microscope. 
For the immunostaining of oxidative stress markers, 4-hydroxynonenal (4-HNE) and 8-hydroxy-2-deoxyguanosine (8-OHdG), 30,31 cryosections were postfixed in acetone and stained using the ABC kit according to the supplier's instructions (Vector Laboratories, Burlingame, CA). 18 Sections were incubated with the following primary antibodies: mouse monoclonal anti-4-HNE (1:400; JaICA, Shizuoka, Japan) and mouse monoclonal anti-8-OHdG (1:20; JaICA), in a blocking buffer for 1 hour at 4°C. The stained sections were further counterstained for nuclei with methyl green (0.5%). A light microscope was used to count the number of 4-HNE- and 8-OHdG-positive cells in each section of the retina at a final magnification of ×200. 
For immunofluorescent staining of microglia and macroglia (astrocytes and Müller cells), cryosections were incubated with the following primary antibodies: rabbit polyclonal anti-Iba1 32 (1:100; Wako, Osaka, Japan) or rabbit polyclonal anti-glial fibrillary acidic proteins (GFAP; 1:500; DAKO, Glostrup, Denmark) in blocking buffer for 1 hour at room temperature. After they were washed twice with PBS, the sections were incubated with fluorescein isothiocyanate (FITC)–conjugated secondary antibody (1:100; Invitrogen, Carlsbad, CA) for 30 minutes and further counterstained for nuclei with propidium iodide for 10 minutes. A laser scanning confocal microscope (FV300; Olympus, Tokyo, Japan) was used to count the number of Iba1-positive cells in each section of the retina at a final magnification of ×200. 
Statistical Analysis
All data are presented as the mean ± SD. For single comparisons, we performed an unpaired two-tailed Student's t-test. For multiple comparisons, we used an analysis of variance (ANOVA) followed by the Fisher least-significant difference (LSD) test (StatView; SAS Institute, Cary, NC). P < 0.05 was considered statistically significant. 
Results
Effect of H2-Loaded Eye Drops on H2 Concentration in the Vitreous Body and the Accumulation of ·OH during Retinal I/R
We prepared an H2-saturated normal saline solution (0.8 mM, pH 7.2; H2-loaded eye drops) and packed it into an aluminum foil bag to prevent a decrease in H2 concentration. A dropper connected to the bag was held close to the rat's eye, and drops were applied to the ocular surface. The time-course of changes in H2 levels was monitored with a needle-shaped hydrogen sensor electrode inserted through the sclera to the vitreous body. When H2-loaded eye drops were administered continuously, approximately 0.5 mM H2 was detected on the ocular surface (Fig. 1C). Two minutes after the start of administration, H2 concentration in the vitreous body started to increase and reached a maximum level after 15 minutes (Fig. 1D). At that time, the H2 concentration accounted for approximately 20% (0.16 mM) of the H2-loaded eye drops. Immediately after administration of the H2-loaded eye drops ceased, the H2 concentration in the vitreous body was observed to gradually decrease and then completely disappear after 15 minutes (Fig. 1D). The maximum observed concentration of H2 in the vitreous body was approximately one third of that observed on the ocular surface (Fig. 1E). 
Figure 1.
 
H2-loaded eye drops increased intravitreal H2. (A) H2-loaded eye drops were prepared by bubbling H2 gas (solid thick line, flow rate: 1 L/min) through 400 mL of normal saline solution. After the bubbling was stopped, the H2 concentration was gradually decreased by stirring and reached <1% within 90 minutes. (B) H2-loaded eye drops were stored in an aluminum foil bag and administered to the ocular surface with a dropper. Scale bar, 4 cm. The concentrations of H2 on the ocular surface (C) and in the vitreous body (D) were monitored with a needle-type H2 sensor. Solid thick line: application times of H2-loaded eye drops (4 mL/min). (E) Summary data showing H2 concentration on the ocular surface (OS; n = 3) and in the vitreous body (VB; n = 3). Data represent the mean ± SD.
Figure 1.
 
H2-loaded eye drops increased intravitreal H2. (A) H2-loaded eye drops were prepared by bubbling H2 gas (solid thick line, flow rate: 1 L/min) through 400 mL of normal saline solution. After the bubbling was stopped, the H2 concentration was gradually decreased by stirring and reached <1% within 90 minutes. (B) H2-loaded eye drops were stored in an aluminum foil bag and administered to the ocular surface with a dropper. Scale bar, 4 cm. The concentrations of H2 on the ocular surface (C) and in the vitreous body (D) were monitored with a needle-type H2 sensor. Solid thick line: application times of H2-loaded eye drops (4 mL/min). (E) Summary data showing H2 concentration on the ocular surface (OS; n = 3) and in the vitreous body (VB; n = 3). Data represent the mean ± SD.
To verify that the diffused H2 protects against ·OH during retinal I/R, we assessed the accumulation of ·OH by the fluorescence signal emitted by the oxidized form of HPF. 28 We produced retinal ischemia in rats by increasing IOP with an infusion needle connected to a saline bag. Just before the induction of ischemia, 4 μL of 1 mM HPF was given intravitreally, followed by 60 minutes of ischemia. Fifteen minutes after reperfusion, the retinas were flatmounted and imaged in their entirety using a laser confocal-scanning microscope (Fig. 2A). The retinal HPF-fluorescence in the H2-loaded eye drop-treated group was significantly less than that in the vehicle-treated group (Fig. 2B). 
Figure 2.
 
H2-loaded eye drops reduced hydroxyl radicals in the retina. HPF was given intravitreally just before the induction of ischemia. After I/R, the retinas were quickly removed and flatmounted. (A) Representative fluorescent images were obtained with a laser scanning confocal microscope. (B) HPF fluorescence was quantified from the entire retina of each independent experiment. *P < 0.01. Data represent the mean ± SD. Scale bar, 200 μm.
Figure 2.
 
H2-loaded eye drops reduced hydroxyl radicals in the retina. HPF was given intravitreally just before the induction of ischemia. After I/R, the retinas were quickly removed and flatmounted. (A) Representative fluorescent images were obtained with a laser scanning confocal microscope. (B) HPF fluorescence was quantified from the entire retina of each independent experiment. *P < 0.01. Data represent the mean ± SD. Scale bar, 200 μm.
Effect of H2-Loaded Eye Drops on the Number of Apoptotic and Oxidative Stress Marker–Positive Cells
To determine whether the administration of H2-loaded eye drops protects against retinal I/R injury, eye drops with and without H2 were applied during the entire 90 minutes process (60 minutes of ischemia followed by 30 minutes of reperfusion). One day after I/R injury, a remarkable increase in the number of apoptotic cells (TUNEL-positive cells) was observed in both the inner and the outer nuclear layers of vehicle-treated retinas (Fig. 3A); however, the administration of H2-loaded eye drops resulted in a significant decrease (approximately 77%, P < 0.0001) of TUNEL-positive cells (Figs. 3A, 3B), indicating that H2-loaded eye drops had potent antiapoptotic activity. We speculate that the decreased apoptotic cell death reflects the H2-dependent reduction of oxidative stress, which was mainly promoted by ·OH. 
Figure 3.
 
H2-loaded eye drops reduced apoptotic cell death and oxidative stress. One day after I/R, the eyes were immediately enucleated and fixed for TUNEL assay (A, B) and staining with antibodies against oxidative stress markers 4-HNE (C, D) and 8-OHdG (E, F). Images of representative slices (A, C, E) and the number of positive cells per field of view (FOV) (B, D, F) in normal retina (N) and the I/R-injured retinas treated with the vehicle (H2 −) or the H2-loaded eye drops (H2 +) are shown (n = 5 animals per group). **P < 0.001. ***P < 0.0001. Data represent the mean ± SD. Scale bar, 30 μm.
Figure 3.
 
H2-loaded eye drops reduced apoptotic cell death and oxidative stress. One day after I/R, the eyes were immediately enucleated and fixed for TUNEL assay (A, B) and staining with antibodies against oxidative stress markers 4-HNE (C, D) and 8-OHdG (E, F). Images of representative slices (A, C, E) and the number of positive cells per field of view (FOV) (B, D, F) in normal retina (N) and the I/R-injured retinas treated with the vehicle (H2 −) or the H2-loaded eye drops (H2 +) are shown (n = 5 animals per group). **P < 0.001. ***P < 0.0001. Data represent the mean ± SD. Scale bar, 30 μm.
We then examined the levels of two oxidative stress markers, 4-HNE and 8-OHdG, in the vehicle-treated and the H2-loaded eye drop–treated eyes by immunohistochemical staining with each specific antibody. 30,31 As expected, 9 1 day after I/R injury, the number of 4-HNE- and 8-OHdG-positive cells increased dramatically in the retina (Figs. 3C, 3E, respectively). However, eyes that had been treated with H2-loaded eye drops exhibited significantly fewer 4-HNE and 8-OHdG-positive cells compared with the vehicle-treated retinas (Figs. 3C–F), supporting our formulated hypothesis. 
Effect of H2-Loaded Eye Drops on Histopathologic and Morphometric Changes
To further evaluate the protective effect of H2-loaded eye drops, we observed histopathologic and morphometric changes 7 days after retinal I/R injury. First, eye drops with and without H2 were applied during the entire 90-minute process (duration F in Fig. 4A). Histopathologic changes of the retina at 7 days after I/R injury are depicted in Figure 4B. The H2-loaded eye drop–treated group showed a nearly normal structure with a thicker retina; however, the H2-free (vehicle) eye drops–treated group exhibited a marked thinning and atrophy of the retina. Quantitative morphometry of retinal thickness was used to estimate the effect of H2 (Fig. 4C). The thickness in the I/R-injured retina treated with the H2-loaded eye drops (102.6 ± 3.8 μm) increased significantly compared with the retina treated with the vehicle (66.9 ± 7.8 μm, P < 0.0001). In normal retina from untreated animals, the mean thickness of the retina was 117.0 ± 4.5 μm, indicating that the H2-loaded eye drops improved the recovery of retinal thickness by >70%. 
Figure 4.
 
H2-loaded eye drops prevented retinal degeneration caused by I/R. One week after I/R injury, the retinas were sliced and stained with H&E. (A) Schematic of the experiment, with four different durations of H2-loaded eye drops administration. (B) Images of representative slices of normal retinas, I/R-injured retinas treated with vehicle, and retinas treated with H2-loaded eye drops during the entire 90-minute process (60 minutes of ischemia followed by 30 minutes of reperfusion) are shown. Scale bar, 50 μm. (C) Retinal thicknesses for different durations of H2-loaded eye drops (100%) administration. ***P < 0.0001 compared with I/R-injured retina treated with the vehicle (H2 −). (D) Retinal thicknesses at different concentrations of H2 in eye drops. The retinas were treated with H2-loaded eye drops during the entire process (duration F). *P < 0.01, **P < 0.001, ***P < 0.0001 compared with I/R-injured retina treated with 0% H2. Histograms represent the mean ± SD.
Figure 4.
 
H2-loaded eye drops prevented retinal degeneration caused by I/R. One week after I/R injury, the retinas were sliced and stained with H&E. (A) Schematic of the experiment, with four different durations of H2-loaded eye drops administration. (B) Images of representative slices of normal retinas, I/R-injured retinas treated with vehicle, and retinas treated with H2-loaded eye drops during the entire 90-minute process (60 minutes of ischemia followed by 30 minutes of reperfusion) are shown. Scale bar, 50 μm. (C) Retinal thicknesses for different durations of H2-loaded eye drops (100%) administration. ***P < 0.0001 compared with I/R-injured retina treated with the vehicle (H2 −). (D) Retinal thicknesses at different concentrations of H2 in eye drops. The retinas were treated with H2-loaded eye drops during the entire process (duration F). *P < 0.01, **P < 0.001, ***P < 0.0001 compared with I/R-injured retina treated with 0% H2. Histograms represent the mean ± SD.
To investigate the effect of different durations of H2-loaded eye drop administration, we applied H2-loaded eye drops using three different time courses (Fig. 4A) and observed that the H2-loaded eye drops exerted their effect only when H2 was already inside the eyeball at the onset of reperfusion (Fig. 4C). There were no significant differences in retinal thickness between groups treated with H2-loaded eye drops only after reperfusion (duration R; 75.4 ± 10.4 μm) and treated with vehicle (P = 0.06). However, the retina that was treated with H2-loaded eye drops only during ischemia (duration I; 91.6 ± 7.5 μm) was still significantly thicker than that treated with the vehicle (P < 0.01). We next administered H2-loaded eye drops for 10 minutes before and 30 minutes after reperfusion (duration I/R) and observed that the administration schedule was sufficient to suppress the reduction of retinal thickness (duration I/R; 96.6 ± 4.4 μm; P < 0.001 vs. vehicle). Furthermore, we applied eye drops diluted to 25%, 50%, and 75% of the normal H2-loaded eye drops during the entire 90-minute process and observed that H2-loaded eye drops suppressed the reduction of retinal thickness in a dose-dependent manner (Fig. 4D). It is notable that the 25%-diluted H2-loaded eye drops were still effective. 
Effect of H2-Loaded Eye Drops on Glial Activation
Considering the critical role of increasing glial activation in the pathogenic progression of retinal damage, we investigated the immunohistochemical changes of the Iba1 32 and GFAP 33 at 7 days after retinal I/R injury with and without H2 treatment. Iba1 is specifically expressed by microglia/macrophages. 34 A small number of Iba1-positive cells was observed in normal retinas, whereas an increasing number of Iba1-positive cells was observed in I/R-injured retinas. At that time, H2-loaded eye drops were observed to inhibit the activation of microglia (Figs. 5A, 5B), indicating that the ongoing neurodegeneration, which activated microglia, was repressed by H2. In addition, H2-loaded eye drops repressed the increase in GFAP immunoreactivity in I/R-injured retinas. The only GFAP-positive cells in normal retina are astrocytes, whereas in the injured retinas, Müller cells, the specific glial cells in the retina, react with anti-GFAP antibody across the retinal layers. 33 In vehicle-treated retinas, GFAP was quite prominent in the Müller cells across the retinal layers and was also strongly present in the astrocytes of the nerve fiber layers, when compared with the H2-loaded eye drop–treated retinas (Fig. 5C). 
Figure 5.
 
H2-loaded eye drops prevented glial activation caused by I/R in the retina. One week after I/R injury, the retinas were sliced, stained with antibodies to Iba1 (a marker for microglia/macrophages, green) (A, B) and GFAP (a marker for Müller cells and astrocytes, green) (C), and further counterstained for nuclei with propidium iodide (red). Images of representative slices (A, C) and the number of positive cells per field of view (FOV) (B) in normal retina (N) and I/R-injured retinas treated with the vehicle (H2 −) or H2-loaded eye drops (H2 +) are shown (n = 5 animals per group). *P < 0.01. Data represent the mean ± SD. Scale bar, 50 μm.
Figure 5.
 
H2-loaded eye drops prevented glial activation caused by I/R in the retina. One week after I/R injury, the retinas were sliced, stained with antibodies to Iba1 (a marker for microglia/macrophages, green) (A, B) and GFAP (a marker for Müller cells and astrocytes, green) (C), and further counterstained for nuclei with propidium iodide (red). Images of representative slices (A, C) and the number of positive cells per field of view (FOV) (B) in normal retina (N) and I/R-injured retinas treated with the vehicle (H2 −) or H2-loaded eye drops (H2 +) are shown (n = 5 animals per group). *P < 0.01. Data represent the mean ± SD. Scale bar, 50 μm.
Discussion
H2-loaded eye drops have a strong protective effect against retinal I/R injury. Previous studies have demonstrated that antioxidants can decrease retinal injury 615 ; however, because antioxidants are difficult to deliver into the vitreous body by topical administration, they were injected into either the eye or the peritoneal cavity. Thus, easily applicable antioxidative reagents without significant side effects are strongly desirable. H2 is an antioxidant that can easily diffuse into the body. We have observed that H2 diffuses into the organelles, including mitochondria and the nucleus, of cultured cells. 18 These properties prompted us to attempt the administration of H2-loaded eye drops for retinal diseases. This is the first report that H2 can immediately penetrate the vitreous body after the administration of H2-loaded eye drops, thereby directly reducing a toxic ROS, ·OH, which is produced during I/R. This effectively protects the retina from I/R injury. 
Although the sources and mechanisms of ROS generation during I/R by transiently raised IOP are not clearly understood, ROS kills neurons in the ganglion cell layer, inner nuclear layer, and outer nuclear layer mainly by apoptosis. 5,9,35 Ophir et al. 14,15 demonstrated that a burst of ·OH occurs in the cat retina during the early reperfusion phase (5 minutes of reperfusion). Thus, we assessed ·OH after 15 minutes of reperfusion with HPF fluorescence and found that the accumulation of ·OH was reduced by H2-loaded eye drops in the I/R-injured retina, indicating that H2 directly reduced ·OH and decreased subsequent oxidative stress. Indeed, 1 day after reperfusion, H2-loaded eye drops dramatically decreased 4-HNE-, 8-OHdG-, and TUNEL-positive cells indicating that H2 protected lipids from peroxidation and DNA from oxidation and reduced subsequent retinal cell death (detected as apoptosis) after I/R injury. 
Neurodegeneration was obvious at 7 days after retinal I/R injury. Previous studies on retinal damage 7 days after I/R injury have shown that the thinning of the retina was evident both morphologically and morphometrically. 1,26,36,37 In the present study, H2-loaded eye drops clearly suppressed the thinning of the retina. However, when H2-loaded eye drops were applied after the onset of reperfusion (duration R), they did not protect from retinal damage (Fig. 4). As shown in Figure 1, H2 concentration in the vitreous body gradually increased after 2 minutes and reached its maximum level after 15 minutes. Immediately after H2-loaded eye drop administration was stopped, the H2 level gradually decreased and then completely disappeared after 15 minutes. Thus, H2 applied after the onset of reperfusion could not reach a level sufficient to inhibit the accumulation of ·OH in the early reperfusion phase, whereas H2 applied before or during reperfusion (duration I or I/R) had a high enough H2 level. 
Microglia, Müller cells, and most likely astrocytes respond within hours to elevation of IOP in the retina. 38 Heterogeneous populations of microglia/macrophages are observed in the normal retina and activated early after I/R injury. 39 Dying neurons are phagocytosed by them. The long duration of ROS production (up to 48 hours after I/R) may be explained partly by the infiltration of microglia/macrophages into the site of inflammation. 9 The presence of GFAP in a glial cell is considered a marker for reactive gliosis, which is not neuroprotective, but rather promotes neurodegeneration. 40 H2-loaded eye drops reduced the number of reactive glia, indicating that H2-loaded eye drops during I/R were sufficient to suppress harmful gliosis after I/R injury and recover the thickness of the retina. 
In conclusion, this study demonstrates that the topical application of H2 can be a useful antioxidant to protect against retinal I/R injury by direct H2 diffusion into the retina. Accordingly, this neuroprotective antioxidant could offer a new therapeutic strategy to the clinical setting to reduce retinal damage in acute glaucoma and acute retinal vascular occlusion. 
Footnotes
 Supported in part by Ministry of Education, Culture, Sports, Science, and Technology of Japan Grants-in-Aid 19659331 (SO) and 20500345 (IO).
Footnotes
 Disclosure: H. Oharazawa, None; T. Igarashi, None; T. Yokota, None; H. Fujii, None; H. Suzuki, None; M. Machide, None; H. Takahashi, None; S. Ohta, None; I. Ohsawa, None
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Figure 1.
 
H2-loaded eye drops increased intravitreal H2. (A) H2-loaded eye drops were prepared by bubbling H2 gas (solid thick line, flow rate: 1 L/min) through 400 mL of normal saline solution. After the bubbling was stopped, the H2 concentration was gradually decreased by stirring and reached <1% within 90 minutes. (B) H2-loaded eye drops were stored in an aluminum foil bag and administered to the ocular surface with a dropper. Scale bar, 4 cm. The concentrations of H2 on the ocular surface (C) and in the vitreous body (D) were monitored with a needle-type H2 sensor. Solid thick line: application times of H2-loaded eye drops (4 mL/min). (E) Summary data showing H2 concentration on the ocular surface (OS; n = 3) and in the vitreous body (VB; n = 3). Data represent the mean ± SD.
Figure 1.
 
H2-loaded eye drops increased intravitreal H2. (A) H2-loaded eye drops were prepared by bubbling H2 gas (solid thick line, flow rate: 1 L/min) through 400 mL of normal saline solution. After the bubbling was stopped, the H2 concentration was gradually decreased by stirring and reached <1% within 90 minutes. (B) H2-loaded eye drops were stored in an aluminum foil bag and administered to the ocular surface with a dropper. Scale bar, 4 cm. The concentrations of H2 on the ocular surface (C) and in the vitreous body (D) were monitored with a needle-type H2 sensor. Solid thick line: application times of H2-loaded eye drops (4 mL/min). (E) Summary data showing H2 concentration on the ocular surface (OS; n = 3) and in the vitreous body (VB; n = 3). Data represent the mean ± SD.
Figure 2.
 
H2-loaded eye drops reduced hydroxyl radicals in the retina. HPF was given intravitreally just before the induction of ischemia. After I/R, the retinas were quickly removed and flatmounted. (A) Representative fluorescent images were obtained with a laser scanning confocal microscope. (B) HPF fluorescence was quantified from the entire retina of each independent experiment. *P < 0.01. Data represent the mean ± SD. Scale bar, 200 μm.
Figure 2.
 
H2-loaded eye drops reduced hydroxyl radicals in the retina. HPF was given intravitreally just before the induction of ischemia. After I/R, the retinas were quickly removed and flatmounted. (A) Representative fluorescent images were obtained with a laser scanning confocal microscope. (B) HPF fluorescence was quantified from the entire retina of each independent experiment. *P < 0.01. Data represent the mean ± SD. Scale bar, 200 μm.
Figure 3.
 
H2-loaded eye drops reduced apoptotic cell death and oxidative stress. One day after I/R, the eyes were immediately enucleated and fixed for TUNEL assay (A, B) and staining with antibodies against oxidative stress markers 4-HNE (C, D) and 8-OHdG (E, F). Images of representative slices (A, C, E) and the number of positive cells per field of view (FOV) (B, D, F) in normal retina (N) and the I/R-injured retinas treated with the vehicle (H2 −) or the H2-loaded eye drops (H2 +) are shown (n = 5 animals per group). **P < 0.001. ***P < 0.0001. Data represent the mean ± SD. Scale bar, 30 μm.
Figure 3.
 
H2-loaded eye drops reduced apoptotic cell death and oxidative stress. One day after I/R, the eyes were immediately enucleated and fixed for TUNEL assay (A, B) and staining with antibodies against oxidative stress markers 4-HNE (C, D) and 8-OHdG (E, F). Images of representative slices (A, C, E) and the number of positive cells per field of view (FOV) (B, D, F) in normal retina (N) and the I/R-injured retinas treated with the vehicle (H2 −) or the H2-loaded eye drops (H2 +) are shown (n = 5 animals per group). **P < 0.001. ***P < 0.0001. Data represent the mean ± SD. Scale bar, 30 μm.
Figure 4.
 
H2-loaded eye drops prevented retinal degeneration caused by I/R. One week after I/R injury, the retinas were sliced and stained with H&E. (A) Schematic of the experiment, with four different durations of H2-loaded eye drops administration. (B) Images of representative slices of normal retinas, I/R-injured retinas treated with vehicle, and retinas treated with H2-loaded eye drops during the entire 90-minute process (60 minutes of ischemia followed by 30 minutes of reperfusion) are shown. Scale bar, 50 μm. (C) Retinal thicknesses for different durations of H2-loaded eye drops (100%) administration. ***P < 0.0001 compared with I/R-injured retina treated with the vehicle (H2 −). (D) Retinal thicknesses at different concentrations of H2 in eye drops. The retinas were treated with H2-loaded eye drops during the entire process (duration F). *P < 0.01, **P < 0.001, ***P < 0.0001 compared with I/R-injured retina treated with 0% H2. Histograms represent the mean ± SD.
Figure 4.
 
H2-loaded eye drops prevented retinal degeneration caused by I/R. One week after I/R injury, the retinas were sliced and stained with H&E. (A) Schematic of the experiment, with four different durations of H2-loaded eye drops administration. (B) Images of representative slices of normal retinas, I/R-injured retinas treated with vehicle, and retinas treated with H2-loaded eye drops during the entire 90-minute process (60 minutes of ischemia followed by 30 minutes of reperfusion) are shown. Scale bar, 50 μm. (C) Retinal thicknesses for different durations of H2-loaded eye drops (100%) administration. ***P < 0.0001 compared with I/R-injured retina treated with the vehicle (H2 −). (D) Retinal thicknesses at different concentrations of H2 in eye drops. The retinas were treated with H2-loaded eye drops during the entire process (duration F). *P < 0.01, **P < 0.001, ***P < 0.0001 compared with I/R-injured retina treated with 0% H2. Histograms represent the mean ± SD.
Figure 5.
 
H2-loaded eye drops prevented glial activation caused by I/R in the retina. One week after I/R injury, the retinas were sliced, stained with antibodies to Iba1 (a marker for microglia/macrophages, green) (A, B) and GFAP (a marker for Müller cells and astrocytes, green) (C), and further counterstained for nuclei with propidium iodide (red). Images of representative slices (A, C) and the number of positive cells per field of view (FOV) (B) in normal retina (N) and I/R-injured retinas treated with the vehicle (H2 −) or H2-loaded eye drops (H2 +) are shown (n = 5 animals per group). *P < 0.01. Data represent the mean ± SD. Scale bar, 50 μm.
Figure 5.
 
H2-loaded eye drops prevented glial activation caused by I/R in the retina. One week after I/R injury, the retinas were sliced, stained with antibodies to Iba1 (a marker for microglia/macrophages, green) (A, B) and GFAP (a marker for Müller cells and astrocytes, green) (C), and further counterstained for nuclei with propidium iodide (red). Images of representative slices (A, C) and the number of positive cells per field of view (FOV) (B) in normal retina (N) and I/R-injured retinas treated with the vehicle (H2 −) or H2-loaded eye drops (H2 +) are shown (n = 5 animals per group). *P < 0.01. Data represent the mean ± SD. Scale bar, 50 μm.
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