Surgical procedures and in vivo experiments

All the procedures used in this study were performed in accordance with the European Union Council Directive for the Care and Use of Laboratory animals, 2010/63/EU and were approved by the local ethics committee of the animal house facilities of the Center for Neurosciences and Cell Biology (ORBEA - Órgão Responsável pelo Bem-Estar Animal). In vivo studies were carried out on adult male Wistar rats (10 weeks) maintained in controlled environmental conditions: temperature of 22–24 °C, relative humidity of 45–65%, 15 air exchanges per hour and a 12:12 light/dark cycle. Animals were housed in filter-topped type III Makrolon cages on an individually ventilated caging system (VentiRack Bioscreen™). Rats were fed with a standard rat chow diet (4RF21-GLP Mucedola, SRL, Settimo Milanese, Italy) and were provided with chlorinated water, both ad libitum.

Rats were anesthetized with urethane (1.25 g/kg, i.p.) and a stereotaxic surgery was performed as previously described²⁹. Animals breathed spontaneously and their body temperature was maintained at 37 °C using a heated pad coupled to a Gaymar Heating Pump (Braintree Scientific, Inc., USA). Basic physiological parameters (blood O₂ saturation, heart rate and breath rate) were continuously monitored during the experiment using a pulse oximeter system (MouseOx^®, Starr life sciences, Oakmont, PA, USA). After exposing the skull, two craniotomies were performed: a primary posterior craniotomy overlying the parietal cortex (2.5 to 4.5 mm posterior and 1.5 to 3.5 mm lateral to bregma) for measurements and a secondary craniotomy (~1 mm², 5 mm posterior to the primary craniotomy) for SD induction. The meninges were removed from the brain surface prior to the insertion of the pre-calibrated LOx-GOx MBA into the rat cerebral cortex according to coordinates calculated from bregma based on the rat brain atlas of Paxinos and Watson⁵⁰: 3.6 mm posterior, 2.0 mm lateral and 1.8 mm ventral to bregma. The LOx-GOx MBA was positioned with the site’s surface in posterior orientation. An Ag/AgCl reference electrode prepared from a silver wire (200 µm diameter) was placed underneath the retracted skin and kept moistened with saline. Electrochemical recordings were performed using the FAST16mkIII high-speed electrochemical system (Quanteon, Nicholasville, KY, USA) applying a constant potential (+0.7 V vs Ag/AgCl) and using a 100 Hz acquisition rate. Cerebral blood flow was simultaneous and continuously measured by laser Doppler flowmetry (Periflux System 5000, Perimed AB, Järfälla, Sweden). The LDF probe (780 nm wavelength, 250 µm fiber separation) was placed on the cortical surface in the immediacy of the MBA. Calibration of the probe was performed routinely according to manufacturer recommendations to equalize the perfusion values among the various recordings. The time constant was set to 0.03 s and the signal-processing unit used a bandwidth of 32 Hz. After a 1 hour stabilization period, SD was induced 5 mm away from the recording site by pricking the upper cortical layers with the tip of a 26-gauge needle as previously described^{6, 8}. As a control experiment, anoxia was induced by forcing the animal to breath pure N₂ gas until cardiac arrest was achieved.