Phenylephrine hydrochloride

Electroretinogram (ERG) were performed on the Diagnosys Espion ColorDome system and manufacturer software (Diagnosys LLC, Lowell, MA, USA) essentially as previously described [19 (link)]. Briefly, mice were anesthetized with 100 mg/kg ketamine and 7.5 mg/kg xylazine. Pupils were dilated with 1% atropine (Falcon Pharmaceuticals, Fort Worth, TX, USA) followed by 2.5% Phenylephrine Hydrochloride (Bausch & Lomb, Portland, OR, USA). A ground electrode was inserted into the tail and a second electrode placed subcutaneously centrally between the eyes as reference. Ophthalmic gel (Gonak, Akorn, Lake Forest, IL, USA) was applied to the cornea and electrodes placed in contact with the gel across each cornea. For light-adapted ERGs, the stimulation was five flashes of 4 ms duration at 1 Hz at 10 cd s/m² with a background of 5 cd s/m².
Dark-adapted ERGs were performed using the protocol previously described [19 (link)]. Briefly, mice were dark adapted overnight and then anesthetized and dilated as described above. A stimulus of ten series of three flashes of light of 4 ms duration was applied with a delay between each series of flashes of 15–60 s (delay period increases with stimulus intensity). The amplitude for the a wave was the lowest point of the initial response relative to baseline. The b wave amplitude was calculated relative to the a wave.

Mice were dark adapted for at least 12 h, and anesthetized by
intraperitoneal injection of 50 mg xylazine and ketamine/kg body
weight. The mouse corneas were anesthetized locally with 0.5%
proparacaine hydrochloride (Bausch & Lomb, Rochester, NY, USA), and the
pupils were dilated with 2.5% phenylephrine hydrochloride (Bausch
& Lomb). The ground and reference electrodes were inserted
subcutaneously in the hind limb and centered along the nasal ridge,
respectively. Gold loop electrodes were placed on each eye. The amplitude of
the a-wave was measured from the baseline to the trough of the a-wave, and
the amplitude of the b-wave was measured from the trough of the a-wave to
the peak of the b-wave. The scotopic ERGs were performed on
IL-1β-treated group and Tn-treated mice after day 10 and
day 30 using LKC Technologies Bigshot Ganzfeld Stimulator, Gaithersburg, MD,
USA, as previously described^{23 (link)} and
were registered with 10 μs flashes of white light at
−20, −10, 0, 5, 10 and 15 db. PBS injection was used as
control for both the treatment groups (IL-1β and Tn).

All animal experiments were performed in compliance with local and national ethical and legal authorities and in accordance with the Association for Research in Vision and Ophthalmology statements on the care and use of animals in ophthalmic research. JAX C57BL/6J mice were purchased from Charles River Laboratories and housed in the Biomedical Sciences Division (University of Oxford, Oxford, UK). Animals were kept in a 12-hr light/12-hr dark cycle, with food and water available ad libitum. All experiments were conducted in 6- to 8-week-old female mice, and surgery and in vivo imaging were performed under general anesthesia. Animals were anesthetized by intraperitoneal injection of ketamine (Vetalar, 80 mg/kg body weight) and xylazine (Rompun, 10 mg/kg body weight) and the pupils fully dilated with tropicamide 1% and phenylephrine hydrochloride 2.5% eye drops (both from Bausch & Lomb). Proxymetacaine hydrochloride 0.5% eye drops were used for additional topical anesthesia. Anaesthesia was reversed following procedures by intraperitoneal injection of atipamezole (Antisedan, 2 mg/kg body weight).

Host mice (5–8 weeks old) were anaesthetized using intraperitoneal ketamine hydrochloride (dose of 80 mg kg⁻¹ body weight) and Xylazine (dose of 10 mg kg⁻¹). Additional local anaesthesia was provided by proxymetacaine hydrochloride eye drops (0.5% m/v minims, Bausch & Lomb). The pupils were dilated using tropicamide (1% w/v minims, Bausch & Lomb) and phenylephrine hydrochloride (2.5% w/v minims, Bausch & Lomb). The cells were transplanted subretinally using a Hamilton syringe and a 34-gauge needle. The animals were recovered using Antisedan (Atipamezole, 2 mg kg⁻¹ bodyweight). At up to 3 weeks post transplantation, the animals were killed and the eyes were enucleated. Following removal of the lens, the eyes were fixed for 1 h in 4% paraformaldehyde in PBS. The tissue was processed as described by cryopreservation in sucrose before embedding in Tissue-Tek O.C.T Compound (Sakura, Alphen aan den Rijn, The Netherlands). Cryosections were cut (18 μm) using a Leica Cryostat and dried before storing at −80 °C.

Rats were anaesthetized with a single 10 mg/ml i.p. injection of Domitor (medetomidine hydrochloride, Pfizer, United Kingdom) and Ketaset (ketamine hydrochloride, Zoetis, NJ, United States). Pupils were dilated with topical applications of 5% (w/v) phenylephrine hydrochloride (Bausch and Lomb) and 0.8% (w/v) tropicamide (Bausch and Lomb), and eyes were coated with Lubrithal (Dechra) to prevent dehydration. A 1.5-cm 34-gauge hypodermic needle (Hamilton, NV, United States) attached to a 5-μl syringe (World Precision Instruments, FL, United States) was inserted through pars plana at 3 mm from the limbus into the vitreous of the left eye at a 45° angle. Rats received 5 μl of sterile PBS, 1 μmol/L NaGYY4137 [sodium 4-methoxyphenyl(morpholino)-phosphinodithioate], or 100 nmol/L AP39 [(10-oxo-10-(4-(3-thioxo-3H-1,2-dithiol-5yl)phenoxy)decyl) triphenylphosphonium bromide] on day 0 (prevention arm) or day 6 (treatment arm).

Animals were obtained from Harlan Laboratories (Shardlow, UK), Charles River (Margate, UK) or in-house colonies and used according to UK Home Office (http://goo.gl/YLUFmF, last accessed 26th October 2015) and the Association for Research in Vision and Ophthalmology Statement for the Use of Animals in Ophthalmic and Vision Research guidelines (http://goo.gl/OwwfLZ, last accessed 26th October 2015). All animals received food and water ad libitum, in a 12-h day/night cycle, temperature-controlled environment.
When required, mice were anesthetized by intraperitoneal (IP) injection of xylazine (0.5 mg/kg) and ketamine (100 mg/kg) in water. Rats were anesthetized by IP injection of 37.5 mg/kg ketamine and 0.25 mg/kg medetomidine hydrochloride. Pupils were dilated with phenylephrine hydrochloride 2.5% and tropicamide 1% (Bausch and Lomb, Surrey, UK) before intravitreal injections.

Mice were dark-adapted overnight, anesthetized with a mixture of Ketamine (133 mg/kg, Streuli, Uznach) and Xylazine (20 mg/kg, Bayler), and pupils dilated with a single eye drop of tropicamide 1% (Théa) and phenylephrine hydrochloride 2.5% (Bausch and Lomb). The animals were kept on a heating pad connected to a temperature control unit to maintain the temperature at 37 °C throughout the experiment. Responses to standard single light flashes (520 nm; half-bandwidth 35 nm) at 0.0001, 0.001, 0.01, 0.03, 0.1, 0.3, 1, 3, 10, and 30 cds/m² for scotopic ERG and after 10 min of light exposure to 3 cds/m² to 0.01, 0.03, 0.1, 0.3, 1, 3, 10 and 30 cds/m² for photopic ERG were generated by a stroboscope (Ganzfeld stimulator, Espion E3 apparatus; Diagnosys LLC). Retinal activity was recorded with a corneal electrode for each eye. The a-wave (photoreceptor-driven first negative wave) amplitude was measured from baseline to the bottom of the a-wave trough, and the b-wave (second order neuron driven, first positive wave) amplitude was measured from the bottom of the a-wave trough to the b-wave peak.