Isoflo vet 100

PND5 and PND9 pups were exposed to neonatal HI as previously described (21 (link), 22 (link)). Pups were anesthetized with isoflurane (IsoFlo vet 100%; Abbott Laboratories Ltd, Illinois, USA), 5% for induction and 1.5% for maintenance, in 1:1 oxygen-nitrogen gas mixture. The left common carotid artery was ligated with a 7.0 silk suture (Ethicon; Vömel, Germany) and the incision was closed and infiltrated with a local anesthetic (Xylocain 20 mg/ml, lidocaine hydrochloride; Astra Zeneca, Södertälje, Sweden). After surgery, mice were returned to their dams for 1 h, before being placed in a chamber with circulating humidified air (36 °C). In the chamber, PND5 mice were exposed to 10 min of air, followed by 60 min of hypoxia (10% O₂ in 90% N₂) followed by another 10 min of air. PND9 mice were treated in the same manner except the hypoxia time was 50 min. This procedure results in a similar degree of brain injury in PND5 and PND9 mice as previously shown (21 (link), 23 (link)) and Supplementary Figure 1. Following the hypoxic exposure, pups were returned to their dams until sacrifice.

Animals were habituated to handling at least 4 days prior to surgery, which was performed during the fourth week. Under general anesthesia, they were implanted with electroencephalography/electromyography (EEG/EMG) electrodes and a microdialysis guide cannula. They were anesthetized with isoflurane (IsoFlo®Vet 100%, Abbott Laboratories Ltd, England) (5% induction, 2% maintenance) and placed in a stereotaxic device. Before the surgery they were injected with buprenorphine (Temgesic®, Indivior UK Limited, Slough, UK, 0.05 mg/kg, s.c.). After exposing, cleaning and disinfecting the skull bone, two gold-coated screws were fitted into the skull for frontal-parietal epidural bipolar recording of the EEG, while two silver wire electrodes were inserted into the neck muscles for EMG recording. A unilateral guide cannula (CMA11 Guide Cannula; CMA/Microdialysis, Stockholm, Sweden) was implanted targeting the basal forebrain cholinergic area (Porkka-Heiskanen et al., 2000 (link)) for subsequent insertion of the microdialysis probe. Electrodes and guide cannula were fixed to the skull with acrylic dental cement. After surgery, animals were individually housed in open Plexiglas boxes, connected to the recording cables through swivels allowing them to move freely. Rats were left for adaptation and recovery for at least 5 days.

The use of animals in this study was carried out under the approval of and in accordance with the regulations of the Animal Laboratory Facility at the Faculty of Medicine at the University of Bergen (accredited by AAALAC International). Albino rats (Wistar HanTac, bred in-house) had ad libitum access to food and water and were kept on a 12/12 light/dark cycle. Two wholemounts from two animals were used for immunolabeling and quantitative analysis. One wholemount was from a 7-week old female rat and one wholemount was from a 4-week old male rat, henceforth referred to as Retina-1 and Retina-2, respectively. The animals were deeply anaesthetized with isoflurane (IsoFlo vet 100%; Abbott Laboratories) in 100% O₂ and killed by cervical dislocation. After removing the eyes, the retinas were dissected out (in HEPES-buffered extracellular solution) and used for immunolabeling.