Atropine

The hamster infection model of SARS-CoV-2 has been described before (15 ). In brief, wild-type Syrian hamsters (Mesocricetus auratus) were purchased from Janvier Laboratories and were housed per two in ventilated isolator cages (IsoCage N Biocontainment System, Tecniplast) with ad libitum access to food and water and cage enrichment (wood block). Housing conditions and experimental procedures were approved by the ethical committee of animal experimentation of KU Leuven (License P065-2020).
Female hamsters 6 wk to 10 wk old were anesthetized with ketamine/xylazine/atropine and inoculated intranasally with 50 µL containing 2 × 10⁶ TCID₅₀. Drug treatment was initiated 1 h before infection. Favipiravir was administered twice daily for 4 d with 300 (oral gavage), 600, or 1,000 mg⋅kg⁻¹⋅d⁻¹ (i.p. injection), starting with a loading dose of 600, 900, or 1,200 mg⋅kg⁻¹⋅d⁻¹, respectively, on the first day. HCQ sulfate (50 mg/kg) was administered once daily by i.p. injection for 4 d. Azithromycin (10 mg/kg) was administered once daily by oral gavage using a 5 mg/mL dilution of Zitromax. Hamsters were daily monitored for appearance, behavior, and weight. At day 4 pi, hamsters were euthanized by i.p. injection of 500 μL of Dolethal (200 mg/mL sodium pentobarbital, Vétoquinol SA), in agreement with the guidelines of the KU Leuven Ethical Committee. Tissues (lungs, small intestine [ileum]) and stool were collected, and viral RNA and infectious virus were quantified by RT-qPCR and end-point virus titration, respectively. Blood samples were collected at day 4 pi for PK analysis of HCQ and favipiravir.

All procedures were approved by the Ethics Committee on Animal Research of the Keio University School of Medicine adhered to the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research, the Institutional Guidelines on Animal Experimentation at Keio University, and the Animal Research: Reporting of In Vivo Experiments (ARRIVE) guidelines for the use of animals in research. C57BL/6 J mice were obtained from CLEA Japan, Inc. Five wild type adult mice without any treatment were used to compare the measurements of AL using μCT and SD-OCT. For lens power dependency experiments, five p21 mice were used for each lens power. For the comparison of LIM with FDM, five p21 mice were worn with 0 D lenses and −30 D lenses, eight p21 mice were worn with −30 D only and ten p21 mice were worn diffusers only. For the atropine treatment experiment, four p21 mice worn 0 D and −30 D lenses were treated with atropine in both eyes and four p21 mice were treated with PBS in both eyes as control. For the OKR experiment, three mice worn −30 D lenses on their right eyes for three weeks started from p21 were used. After the measurement of the OKR, the same three mice were put into general anesthesia for the ERG measurements. All mice were fed with normal chew and water ad lib. Four or five mice with or without the frame were kept in one cage with approximately 50 lux background fluorescent lamp light for 12 h from 8:00 am to 8:00 pm.

All mice were handled for 1 h per day for two weeks prior to the behavioral tests. The behavioral session was conducted in a fear conditioning box (46001, UGO BASILE S.r.l, Italy). Freezing was defined as a complete absence of movement, except for respiration. Scoring of the freezing response duration was started after one second of sustained freezing behavior [34 (link)]. The freezing was measured using the Anymaze (version 6.0, Stoelting) software based on a threshold of change in video image pixels. The mice were delivered with 10 kHz and 1 kHz tones and the percentage of freezing time was averaged for the tone each day.
The changing index of freezing was calculated using the equation: $Thechangingindex=\left(T_{lightonfreezing}-T_{lightofffreezing}\right)/\left(T_{lightonfreezing}+T_{lightofffreezing}\right).$
The discrimination index of fear memory was calculated using the equation: $Thediscriminationindex=\left(T_{10kHzfreezing}-T_{1kHzfreezing}\right)/\left(T_{10kHzfreezing}+T_{1kHzfreezing}\right).$
At the encoding stage (day 1), after 5 min of habituation in context A, the mice received 5 pairings of auditory tone (70 dB SPL 10 kHz or 1 kHz pure tone, 20 s duration) with electric foot shocks (1 mA, 2 s duration, and overlapped with the last 2 s of tone). The time interval between each trial was 70 s. In Fig. 1E, F, and S1C, 10 min before the test, 0.5 µl of pharmacological agent was administered simultaneously bilaterally at a rate of 0.125 µl every 10 s by cannula. The mice were administered either saline, mecamylamine (MCM, 10 µM, 20 µM Aladdin, 826-39-1), or atropine alone (10 µM, 20 µM Aladdin, 5908-99-6). In Fig. 2, each tone is accompanied by optogenetic inhibition or activation. In Fig. 2G, 30 min before the test, mice were intraperitoneally injected with saline and nicotine (10 µM, Sigma Aldrich), and each tone is accompanied by optogenetic inhibition.
At test 0 (day 2), to label tone-responsive ACx neurons, the mice were delivered three times of 10 kHz tone or three times 1 kHz tone.
At test 1a or test 1b (day 2 or day 3), the mice were tested for freezing behavior in response to tests 1a (new fear-unpaired 1 kHz or 10 kHz tone) and 1b (fear-paired 10 kHz or 1 kHz) with a time interval of 2 h in different contexts B and C. The mice were delivered three (Figs. 1, 2, 4, 5 and Figs. S1, 2G, H, 4) or four (Fig. 3, S2E, F) representations of both 10 kHz and 1 kHz tones. In groups with four representations of tones, the first and third tones were accompanied by optogenetic activation or inhibition. In Fig. S2A–C, the mice were only given 10 times optogenetic activations. For test 1, each group of mice was randomly assigned and then equally received 1 kHz or 10 kHz tone in different contexts B and C, and after 90 min, the mice were carefully perfused.
All contexts were different in shape, background, and floor texture.