Vitamin a eye cream

For surgery, animals were anesthetized with a mixture of fentanyl (0.05 mg/kg bodyweight; Sintenyl; Sintetica), midazolam (5 mg/kg bodyweight; Dormicum, Roche), and medetomidine (0.5 mg/kg bodyweight; Domitor; Orion Pharma), administered intraperitoneally. Anesthesia was maintained with midazolam (5 mg/kg bodyweight), injected subcutaneously 50 min after induction. To prevent hypoxemia, a face mask provided 300 ml/min of 100% oxygen. During two-photon imaging mice were anesthetized with 1.2% isoflurane (Attane; Piramal Healthcare, India) and supplied with 300 ml/min of 100% oxygen. Core temperature was kept constant at 37°C using a homeothermic heating blanket system (Harvard Apparatus) during all surgical and experimental procedures. The animal’s head was fixed in a stereotaxic frame (Kopf Instruments) and the eyes were kept moist with ointment (vitamin A eye cream; Bausch & Lomb).

For all experiments, C57BL/6j mice (Charles Rivers, no. 028), three to five months old were used. Both male and female mice were used in the imaging experiment. The mice had free access to water and food and an inverted 12-h light/dark. For the implantation of cranial windows, animals were anesthetized via intraperitoneal injection using a combination of fentanyl (0.05 mg/kg of body weight; brand name: Sintenyl, manufactured by Sintetica), midazolam (5 mg/kg of body weight; brand name: Dormicum, manufactured by Roche), and medetomidine (0.5 mg/kg of body weight; brand name: Domitor, manufactured by Orion Pharma). To ensure proper respiration, a facemask (Narishige International, Japan) was employed, delivering 100% oxygen at a flow rate of 300 ml/min. During 2P imaging, anesthesia was initiated with 4% Isoflurane (Abbott, Cham, Switzerland) in an oxygen/air mixture (100/400 ml/min) and then maintained at 1.2% using a continuous supply of 300 ml/min of 100% oxygen. Throughout all surgical and experimental procedures, the animals’ core temperature was maintained at a steady 37 °C using a homeothermic blanket heating system provided by Harvard Apparatus. The animals’ heads were securely positioned in a stereotaxic apparatus, and their eyes were kept moistened with vitamin A eye cream (Bausch & Lomb)^{69 (link)}.