Medetomidine hydrochloride

Transplantation was conducted as we have described previously (Pearson et al, 2012 (link)) All recipients were adult (6–8 weeks) at time of transplantation. Briefly, recipient mice were anaesthetized with a single intra‐peritoneal injection of a mixture of Dormitor (1 mg/ml medetomidine hydrochloride, Pfizer Pharmaceuticals, Kent, UK), ketamine (100 mg/ml, Fort Dodge Animal Health, Southampton, UK) and sterile water for injections in the ratio of 5:3:42. Prior to cell transplantation, the tip of 34‐G needle was inserted to the sclera and slowly removed. The needle with the cell suspension was then inserted tangentially in through the sclera and RPE, injecting slowly the 1 μl cell suspension, or 2 µl (2 μg) of concentrated EV fractions, or 2 μl (2 μg) of Cre protein (ab134845) underneath the RPE in the superior retina resulting in small retinal detachment.

All surfaces were wiped with 70% ethanol prior to surgery. 3 month old rd1/Foxn1^nu mice were anesthetized by intraperitoneal injection (10mL/Kg) of a mixture of a mixture of Dormitor (1 mg/ml medetomidine hydrochloride; Pfizer Pharmaceuticals), ketamine (100 mg/ml; Fort Dodge Animal Health), and sterile water in the ratio 5:3:42. Tropicamide (1%; Bausch & Lomb) was used to dilate the pupils and topical anesthetic was applied (Tetracaine). Eyes were kept moist by using Viscotears (Novartis Pharmaceuticals UK Ltd). Surgery was performed under direct visual control using an operating microscope (Zeiss). A sterile 34-gauge hypodermic needle was used to make a small puncture to the anterior chamber to relieve pressure in the orbit. The same needle was used to make a new transcleral entry in the posterior orbit and slowly inject 1.5μl of cell suspension into the sub-retinal space. The same region of the eye was targeted for all injections. Significant care was taken not to rupture the very thin remaining neural retina and the needle was left in place for ∼20 s to allow for re-equilibration of intraocular pressure before slowly withdrawing. Anaesthesia was then reversed using same amount of Antisedan, 10mL/Kg, (atipamezole hydrochloride 0.10 mg/ml, Pfizer Pharmaceuticals, Kent UK), with the mice placed on heat mats until fully recovered.