30 gauge needle

The injections were performed under general anesthesia with isoflurane inhalation. The hairs on the back were shaved using hair clippers, followed by hair removal with a depilatory cream (Epilat; Kracie Pharma, Tokyo, Japan). Each test substance was injected onto the parietal periosteum (0.2 mL), intradermally in the left scapular area (0.1 mL), and subcutaneously in the left lumbar area (0.5 mL) (Figure 6). As a negative control, the same volumes of phosphate-buffered saline (PBS) were injected on the right side in the same manner. A 1-mL Luer-lock syringe (TOP Corporation, Tokyo, Japan) with a 30-gauge needle (NIPRO Corporation, Osaka, Japan) was used to inject the test substance. A 6-0 nylon thread was used to make a landmark of each injection site. The rats were maintained under a 12-h day/night cycle and were fed standard feed with water ad libitum. At the end of each experimental period (4 weeks, short-term experiment; 24 weeks, long-term experiment), the rats were euthanized by anesthetic overdose. Assuming clinical use, the injections were given to three different layers and the injection volumes were determined as the minimum amount, with which the swelling could be confirmed at each layer: 0.2 mL on the periosteum, 0.1 mL intradermally, and 0.5 mL subcutaneously.

Aqueous humor was aspirated before IAI and cataract surgery under topical anesthesia using a syringe with a 30-gauge needle (Nipro, Osaka, Japan) by the same procedure. The aqueous humor samples were immediately mixed with 2 μL of protease inhibitor cocktail (Sigma, St. Louis, MO, USA) to prevent complement and anaphylatoxin activation. Aliquots of the obtained aqueous humor samples were stored at − 80 °C until analysis.