Floxal

Wistar rats received an intraocular S100B injection (n = 6–7/group). Before intraocular treatment, animals were anesthetized with a mixture of ketamine (50 mg/mL, Ratiopharm, Ulm, Germany) and xylazine (2%, Bayer Health Care, Leverkusen, Germany) followed by a topical anesthetic (Conjuncain, 4 mg/mL, Bausch & Lomb, Rochester, NY, USA) and a mydriatic to dilate the pupil (Tropicamide, 5 mg/mL, Pharma Stulln, Stulln, Germany). In the S100B group, 0.4 µg/µL S100B solution (Sigma-Aldrich) was injected in the vitreous of one eye with a 32-gauge needle (Hamilton, VWR, Radnor, PA, USA) under a stereomicroscope (Zeiss, Jena, Germany) [29 (link)]. Controls received 2 µL phosphate-buffered saline (PBS; Biochrome), which also functioned as a solvent for S100B. After the injection, the eyes were treated with an antibiotic ointment (Floxal, Bausch & Lomb). Animals were examined 2 h after injection and on the next day. Once a week, the general behavior and look of the animals was examined Eye bleeding or cataracts were considered as early endpoints in this study.

The experiments were performed on 8-week old male Wistar rats, weighing approximately 200 g. All animals were provided by the Center for Experimental Medicine, Medical University of Silesia, Katowice, Poland and were treated in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research and the EC Directive 86/609/EEC for animal experiments, using the protocols approved and monitored by the Local Committee for Animal Experiments of Medical University of Silesia.
All surgical procedures were performed under general anaesthesia with an intraperitoneal injection of a mixture of ketamine (50 mg/kg, VetaKetam, Vetagro, Poland) and xylazine (5 mg/kg, Xylapan, Vetoquinol Biowet, Poland). For the local anaesthesia, we used 0.5% Proxymetacaine hydrochloridum (Alcaine, Alcon, Fort Worth, TX, US). During the recovery from anaesthesia, the rats were placed in their cages and an ointment containing ofloxacin (Floxal, Bausch&Lomb, Bridgewater, NJ, US) was applied on the cornea to prevent corneal desiccation and infection. After follow-up time, the animals were sacrificed with an intraperitoneal overdose of a mixture of ketamine and xylazine.

The intravitreal injections of HSP27 or PBS were performed as previously described [34 (link)]. Briefly, rats were anesthetized with ketamine (50 mg/mL, Ratiopharm, Ulm, Germany) and xylazine (2%, Bayer Health Care, Leverkusen, Germany). After the pupil was dilated with a mydriatic (Tropicamid 5 mg/mL, Stulln, Germany), a topical anesthetic (Conjuncain 4 mg/mL, Bausch&Lomb, Berlin, Germany) was applied on the eye. The commercially available HSP27 protein (cat. HSP0503; Lot: 097102, AtGen, Yatap-dong, South Korea) was already dissolved in 20 mM Hepesl buffer (pH 7.5). However, to obtain a protein solution of 0.2 µg/µL, we diluted the HSP27 protein in PBS. One eye per animal (n = 12) was injected with 2 µL of 0.2 µg/µL (final concentration: 0.4 µg) HSP27 solution under a stereomicroscope (Zeiss, Oberkochen, Germany), with a 32-gauge needle (Hamilton, Reno, NV, USA). Control animals received the same volume of PBS (Biochrome, Berlin, Germany; n = 12), since this was used as a solvent for HSP27. After the injection, the eyes were treated with an antibiotic ointment (Floxal, Bausch&Lomb, Berlin, Germany). No injection was performed on the contralateral eye.

Mice were anesthetized with ketamine (120 mg/mL, Ratiopharm, Ulm, Germany) and xylazine (16 mg/kg, Bayer Health Care, Leverkusen, Germany). Then, a topical anesthetic was applied onto the eye (Conjuncain, 4 mg/mL Bausch&Lomb, Rochester, NY, United States) followed by a mydriatic to dilatate the pupil (Tropicamide, 5 mg/mL, Stulln, Stulln, Germany). The HSP27 protein (cat. HSP0503; Lot: 097102, AtGen, Yatap-dong, South Korea) was already dissolved in 20 mM phosphate-buffered saline (PBS, pH 7.5). One eye per animal was injected with 1 μL of 0.6 μg/μL HSP27 solution under a stereomicroscope (Zeiss, Oberkochen, Germany) with a 32-gauge needle (Hamilton, Reno, NV, United States). Control animals received the same volume of PBS (Biochrome, Berlin, Germany) since this was used as a solvent for HSP27. After the injection, an antibiotic ointment was dripped on the eye (Floxal, Bausch&Lomb). Non-injected contralateral eyes served as naive controls. After injection, the animals were monitored at close intervals to make sure that they were in good condition. Four weeks after injection, subsequent analyses were performed.

A monoclonal antibody against the complement factor C5 (BB5.1; Hycult Biotech, Uden, Netherlands) was injected intravitreally into one eye of some ONA immunized rats, as previously described (Reinehr et al. 2019a (link)). For this, either a lower (15 μmol, n = 6; ONA + C5-I) or a higher (25 μmol, n = 6; ONA + C5-II) concentration of the antibody was applied. The control group was not treated intravitreally (n = 6). The anti-C5 therapy was first administered 1 day before the first immunization with ONA and was repeated every 2 weeks (Fig. 1).
For the injections, rats were anesthetized with a mixture of ketamine and xylazine (100/4 mg/kg). Mydriasis was induced by tropicamide 5% eye drops, and the ocular surface was locally anesthetized with oxybuprocaine-containing eye drops (Conjuncain, Bausch+Lomb, Rochester, NY, USA). The injection of either 5 or 8 μl BB5.1 was administered into the right eye of the rat under a stereomicroscope (Zeiss, Jena, Germany) using a 32 Gauge Hamilton cannula (Hamilton Company, Reno, NV, USA). An antibiotic eye ointment (Floxal, Bausch+Lomb) was applied onto the treated eye. The untreated left eyes of the animals constituted the ONA group (n = 6).