Animals
Eight-week-old male Wistar rats weighing 200 g were obtained from Sankyo Laboratory Service (Tokyo, Japan). All animal-based experiments were conducted in compliance with the Experimental Animal Ethics Review Committee of Nippon Medical School (approval number: 29-055). Rats were housed in a specific pathogen free environment with a 12 hours light / 12 hours dark cycle. Water and food were available and continuous clinical care (24 hours per day / 7 days per week) was provided throughout the experiment to ensure prompt intervention when needed. All procedures conformed with the guidelines of the Association for Research in Vision and Ophthalmic and Visual Research.
Alkali burn model and preparation of H2-dissolved saline
One eye of each rat (n=104 in total) was subjected to an alkali burn under 3.5% isoflurane inhalation anesthesia, according to the following protocol: a circular piece of filter paper (diameter: 3.2 mm) soaked in 1N NaOH was placed on the central cornea for 1 minute. The corneas were rinsed via dripping (10 ml/min) with physiologic saline (saline group: n=39) or H2-dissolved saline (H2 group: n=39) for 5 minutes prior to alkali exposure, and again after alkali exposure. In each rat, the uninjured normal cornea was used as a control.
H2-dissolved saline was prepared as described in our previous report [7]. Briefly, commercially available saline plastic bags (Otsuka Pharmaceutical, Tokyo, Japan) were placed in an acrylic vacuum chamber filled with H2 gas for 24 hours (Fig 1a). Prior to use, the dissolved concentration of H2 in each bag was confirmed using a needle-type H2 sensor (Unisense, Aarhus, Denmark). In all experiments, H2-dissolved saline was used within 5 minutes after retrieval from the vacuum chamber.
Rats were euthanized by exsanguination under 3.5% isoflurane inhalation anesthesia at 6 hours, 1 day, and 7 days after alkali injury. The enucleated eyes were subjected to histological and immunohistochemical examination and low-vacuum scanning electron microscopy (LV-SEM). No adverse events were noted throughout the experiment.
Histological and immunohistochemical analysis and LV-SEM observation
The enucleated eyes (n= 8 for each group / for each endpoint) were fixed in 10% buffered formalin and embedded in paraffin prior to light microscopic observation. Subsequently, deparaffinized tissue sections (thickness: 2.5 μm) were stained with hematoxylin and eosin (HE), Naphtol AS-D chloroacetate esterase (EST) to detect infiltrating neutrophils [14], and LV-SEM. For the latter, tissues were stained with periodic acid-methenamine silver (PAM) to specify collagens [15,16].
For the immunohistochemical analysis of inflammatory cells, neovascularization, and SOD1 enzyme levels, the number of positive cells per sample was measured pathologically in high-power fields (magnification: 400X) located in two corneal areas (center: 3 fields, periphery: 2 fields). The following primary antibodies were used: 1) monoclonal mouse anti-aminopeptidase P antibody (JG12; Thermo Scientific, Rockford, IL, USA) to detect vascular endothelial cells [17], 2) monoclonal mouse anti-8-OHdG antibody (JaICA, Shizuoka, Japan) to detect DNA oxidative stress [18], 3) monoclonal mouse anti-CD68 antibody (ED1; BMA, Nagoya, Japan) to detect pan-macrophages, 4) monoclonal mouse anti-CD163 antibody (ED2; BMA, Nagoya, Japan) to detect M2 macrophages, and 5) polyclonal rabbit anti-SOD1 antibody (Stressgen, Victoria, BC, Canada) to detect SOD1 enzyme.
As noted above, samples for LV-SEM were subjected to PAM staining for contrast enhancement. After staining with PAM, specimens lacking a mounting cover glass were examined via LV-SEM (Hitachi Tabletop Microscope TM3030, Hitachi High-Technologies Corp., Tokyo, Japan) [19,20]. Ultrastructural alterations in the corneal wound were assessed using an acceleration voltage of 15 kV under 30 Pa for the backscattered electron detector.
Statistical analyses
All results are expressed as means ± standard deviations. The statistical analysis was performed using an analytical software program (Excel; Microsoft, Redmond, WA, USA). Student’s t-test was used to evaluate comparisons. The results were considered statistically significant when *p value < 0.05; **p value < 0.01.