Nod 10

The SCR were euthanized by injecting a lethal dose of pentobarbital, and the lenses were removed and homogenized in saline on ice. The homogenates were centrifuged at 20,400× g for 15 min at 4 °C, and the supernatants were used as samples. A flow-through spectrophotometer (NOD-10, Eicom, Kyoto, Japan) was used to measure the nitric oxide (NO) levels [29 (link)], which were expressed as the level of the NO₂⁻ metabolite. The calpain activity was measured at 505 nm by a Calpain Activity Fluorometric Assay Kit according to the manufacturer’s instructions and represented as the ratio of calpain activity levels in SCR aged 6 weeks. The LPO levels were analyzed by measuring the lipid peroxidation products 4-hydroxynonenal and malondialdehyde using an LPO Assay Kit [30 (link)], and the Ca²⁺-ATPase activity was calculated as the difference in the Pi liberated from ATP measured in the presence and absence of Ca²⁺ [30 (link)]. A Ca Test Kit was used to measure the Ca²⁺ content in the lenses [31 (link)]. The protein levels were determined using a Bio-Rad Protein Assay Kit, and the protein was used to evaluate the LPO levels and Ca²⁺-ATPase activity.

The rats were killed under deep isoflurane anesthesia, and the gastric, jejunal, and ileal mucosas were excised and homogenized in saline on ice. The homogenates were then centrifuged at 20,400× g for 10 min at 4 °C, and the supernatants were collected. The NO levels were measured using ENO-20 (Eicom, Kyoto, Japan) according to our previous study [5 (link),6 (link)]. Briefly, ions of nitrous acid (NO₂⁻) and nitrate (NO₃⁻) were separated on a NO-PAK column (4.6 × 50 mm, Eicom, Kyoto, Japan), and NO₂⁻ was measured with Griess reagent and a NOD-10 (Eicom, Kyoto, Japan) at 540 nm. The levels of NO₂⁻ metabolite produced from NO were expressed as NO levels.

Owing to its instability, the quantitative analysis of NO production in/around the skin wounds was performed by simultaneously measuring the NO metabolites of nitrite (NO₂⁻) and nitrate (NO₃⁻) in the microdialysate. The method for measuring NO₂⁻ and NO₃⁻ levels was described previously47 (link). Briefly, 10 μL of the microdialysate fraction was injected into an ENO-30 NO-detector (Eicom, San Diego, CA). The NO₂⁻ and NO₃⁻ in the microdialysate were separated by a reverse-phase separation column (NO-PAK, 4.6 × 50 mm, Eicom, San Diego, CA), and NO₃⁻ was reduced to NO₂⁻ in a reduction column (NO-RED, Eicom, San Diego, CA) at 35 °C column temperature. Then, the NO₂⁻ was reacted with Griess reagent, which was delivered at a rate of 0.1 mL/min, to form a purple azo dye in a reaction coil at 35 °C. The absorbance of the product dye was detected at 540 nm by a flow-through spectrophotometer (NOD-10, Eicom, San Diego, CA). The mobile phase was purchased from Eicom and delivered at a rate of 0.33 mL/min. The concentrations of NO₂⁻ and NO₃⁻ in the microdialysate were obtained using a standard curve prepared from known concentrations of sodium nitrite and sodium nitrate. The resulting values were converted into percentages, taking the values for samples from diabetic rats without skin wounds as 100%. All experiments were performed with n = 6.