Lacri lube

A chronic model of experimental glaucoma was generated in rats by a producing an increase of IOP using microbead injections (Fluospheres, 10 μm) as described previously^{2 (link)}. Weekly injections into the anterior chamber of eye were made for a sustained increase in IOP for 2 months following which the tissues were harvested. Microbeads were administered using a 25-μL syringe connected to a disposable 33-gauge needle (TSK Laboratory, Japan). All injections were performed under an operating microscope (OPMI Vario S88, Carl Zeiss, Germany). Care was taken to avoid needle contact with the iris or lens and minimise damage. The needle was inserted beneath the corneal surface and 5 μL of beads were injected. Rodents were anesthetized using ketamine (75 mg/kg) and medetomidine (0.5 mg/kg) intraperitoneally^{69 (link)}, and anaesthesia reversed using atipamazole (0.75 mg/kg injection. 0.3% Ciprofloxacin drops (0.3%) (Ciloxan; Alcon Laboratories, Australia) and 0.1% dexamethasone eye drops (Maxidex, Alcon Laboratories) were instilled in the eyes. Lacrilube; (Allergan) was applied to cornea to protect the rodent eyes. IOP was regularly measured with the help of rebound tonometer (Icare Tonovet, Finland). Three consecutive pressure readings were recorded from the eyes and their average taken to determine the intraocular pressure.

Three-month-old AP-2β NCC KO mutants and their control littermates were weighed and anesthetized with an i.p. injection of 2.5% avertin at 0.015 ml/g body weight. LACRI-LUBE (Allergan Inc., Markham, ON, Canada) was applied to the eyes to maintain a moist tear film and whiskers were trimmed so there was no impediment of the probe. A validated commercial rebound tonometer (TonoLab, Vantaa, Finland) was mounted on a retort stand with the probe aligned horizontally and perpendicularly to the central cornea, with the tip positioned at 2-3 mm from the eye as previously advised (Chatterjee et al., 2013 (link)). A mean of six measurements was determined for each eye and this was repeated ten times in order to calculate an overall mean. Measurements were only accepted if they registered as TonoLab readings with the best reproducibility (no bar symbol) or the next best reproducibility (bar present at the bottom of the screen) as per the manufacturer's manual. All measurements were taken between 13:00 to 15:00 in the afternoon and 3 min after injection of avertin, as this has been previously shown to be a period of stable IOP (Ding et al., 2011 (link)).

4T1 cells were harvested as above and 2 × 10⁵ live cells injected in 100 µl of PBS into the 4^th right inguinal mammary fatpad of recipient mice. On day 14 post-inoculation, tumors were surgically removed, at approximately 300 mm³. Mice were dosed s.c. with 0.05 ml/25 g of Buprecare (AnimalCare) 15 min prior to being anesthetized using isoflurane (4% in 100% oxygen at a flow rate of 4 L/min for induction and 2.5 L/min for maintenance). Mice were placed on a heat pad with integrated nasal airflow and their eyes protected from drying by Lacrilube (Allergan). The incision was injected with the local analgesic Marcaine and closed using coated Vicryl needle (Ethicon). Mice received 200 µl saline s.c. and were placed in a heated cage and monitored until they recovered from anesthesia. Two further doses of 0.05 ml/25 g Buprecare were administered 6-8 h post-surgery and the following day. Mice were fed mash diet following surgery and during CTX treatment. The day post-surgery, mice were allocated to treatment groups by normalizing tumor size prior to surgical resection (day 13 measurements) across groups. Mice were monitored for tumor regrowth at the orthotopic site and culled by a Schedule 1 method 2-4 weeks post-surgery and macroscopic lung nodules were counted by an investigator blinded to treatment group.

Anesthesia was induced with 5% isoflurane that was reduced to 1.0–2.5% isoflurane for surgery. After the initial induction of anesthesia, 2.5 mg/kg chlorprothixene was administered (i.p.). Ophthalmic ointment (Lacri-lube, Allergan) was applied to the eyes prior to surgery and removed immediately prior to imaging. Throughout the surgery, body temperature was maintained via a heating pad. The scalp was resected over the right visual cortex and a 4-mm craniotomy performed, exposing the cortex. For ISOI experiments, physiological saline was added to cover the cranial window and mice were then transferred to the ISOI rig. For calcium imaging experiments, a 1-mm glass coverslip was placed over the craniotomy and saline added before transferring the mouse to the ISOI rig.