Tropicamide

The mouse laser-induced CNV model was generated as previously described (22 (link)). Mice were anesthetized by ketamine/xylazine intraperitoneal injection and pupils were dilated with topical 1% tropicamide (Santen, Osaka, Japan). At the time of laser exposure, the rupture of Bruch’s membrane was confirmed by bubble formation. Four laser lesions were induced in four quadrants of each eye at approximately the same distance around the optic nerve using a slit lamp delivery system and a glass coverslip as a contact lens. Laser photocoagulation shots that cause hemorrhage or fail to produce a bubble were excluded from calculation. In the animal experiments, the examiner responsible for CNV model establishment was blinded to the animal grouping.

Isoflurane, benzophenone, mannitol (D-mannitol), a Cholesterol E-Test Kit, and DDC were purchased from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). DIS powder and 2-hydroxypropyl-β-cyclodextrin (HPCD) were kindly donated by Ouchi Shinko Chemical Industrial Co., Ltd. (Tokyo, Japan) and Nihon Shokuhin Kako Co., Ltd. (Tokyo, Japan), respectively. A Mitochondrial Isolation kit, a Cytochrome c Oxidase Assay kit, and an ATP Bioluminescent Assay kit were purchased from Sigma Aldrich Japan (Tokyo, Japan). A Bio-Rad Protein Assay kit was provided by BIO-RAD (California, USA). We obtained 0.5% phenylephrine and 0.5% tropicamide from Santen Pharmaceutical Co., Ltd. (Osaka, Japan). An ELISA Insulin Kit and an RNA PCR Kit (AMV Ver 3.0) were purchased from Morinaga Institute of Biological Science Inc. (Kanagawa, Japan) and Takara Bio Inc. (Shiga, Japan), respectively. Methylcellulose type SM-4 (MC) was obtained from Shin-Etsu Chemical Co., Ltd. (Tokyo, Japan). Pentobarbital was provided by Sumitomo Dainippon Pharma Co., Ltd. (Toyo, Japan). Benzalkonium chloride (BAC), methanol, and acetonitrile was purchased from Kanto Chemical Co., Inc. (Tokyo, Japan). LightCycler FastStart DNA Master SYBR Green I was provided by Roche Diagnostics Applied Science (Mannheim, Germany). All other used chemicals were of the highest purity commercially available.

All animal experiments followed the guidelines of the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research and were approved by the Animal Care Committee of Kansai Medical University (approval number: 17–013). Wild-type (WT) C57BL/6J mice were purchased from CLEA Japan (Tokyo, Japan). A total of 10 female mice aged 6 weeks were used for this study. All mice were kept in pathogen-free plastic cages with 12 hour light-dark cycles and had continuous free access to water and food. All plastic cages, water and bedding feed were purified before use. For all procedures, anesthetization was achieved by intraperitoneal injection of 90 mg/kg ketamine hydrochloride (Daiichi Sankyo Co., Tokyo, Japan) and 40 mg/kg xylazine (Bayer, Berlin, Germany); pupils were dilated with topical 0.5% tropicamide and 0.5% phenylephrine (Santen Pharmaceutical, Osaka, Japan).

Ophthalmological examination of all infected animals was carried out with pupils dilated with tropicamide (Santen Pharmaceutical, Japan) and corneas protected by Gatifloxacin Ophthalmic Gel (Shenyang Xingqi eye medicine Co., Ltd) under anesthesia with sodium pentobarbitone (50 mg/kg, Shanghai Hailing Biotechnology Co., Ltd). Comprehensive ocular examinations were performed as follows: the anterior segment was examined by portable slit lamp, and the fundus was examined by MICRON IV retinal microscope (Phoenix Micron IV, Phoenix Research Labs, CA, USA); retinal vascular system was examined using MICRON IV retinal microscope after intraperitoneally injection with 2% fluorescein sodium (Health Manufacturing Services B.V., USA). After ocular observations, the animals were placed on the heat preservation pad for resuscitation, and then tobramycin eye ointment was applied to both eyes. The animals were put into cages after they were fully awake. All animals were observed and recorded, and the observation interval was every 2 h within 3 dpi, every 2 days between 4 and 14 dpi and every 1 week after 14 dpi. According to previous literature, the migration of Toxocara larvae in paratenic hosts can be divided into three phases, namely the acute, subacute and chronic phases [25 (link)]. The observation period was 4 months for C57BL/6 mice and 2 months for gerbils and rats.

The animals were cared for in accordance with the Guide for the Care and Use of Laboratory Animals published by the United States National Institutes of Health. The protocols were also approved by the Institutional Animal Care and Use Committee (IACUC) of Chung-Ang University (IACUC number: 201800044, start date: 4 May 2018). Nine- to ten-week-old male C57BL/6 mice were purchased from Orient Co., Ltd. (Seoul, Korea). Mice were housed in microisolator cages on individually ventilated cage racks with ad libitum access to an autoclaved standard rodent diet (LabDiet 5008, Purina, St. Louis, MO, USA) and were kept under a 12:12 h light/dark cycle. Anesthesia was performed via an intraperitoneal injection of ketamine hydrochloride (100 mg/kg body weight) and xylazine hydrochloride (6 mg/kg body weight). The pupils of the anesthetized mice were dilated with topical drops of 1% tropicamide (Santen, Osaka, Japan).