Measuring Cerebral Blood Flow Responses

Anesthesia was initiated with isoflurane (induction: 5%, maintenance: 2%) and maintained with 50 mg/kg of α-chloralose i.p. (MilliporeSigma, Oakville, ON, Canada) and 750 mg/kg of urethane i.p. (MilliporeSigma, Oakville, ON, Canada). The depth of anesthesia was checked by testing corneal reflexes and motor responses to tail pinch. Mean blood pressure and blood sample collection for gas assessment were monitored through the catheterization of the femoral artery. Mice were artificially ventilated with a nitrogen/oxygen/CO₂ mixture through tracheal intubation. Body temperature was maintained at 37 °C throughout the experiment. CBF was monitored with a laser Doppler probe (AD Instruments, Colorado Springs, CO, USA) placed on the thinned skull above the whisker-barrel area of the somatosensory cortex. The flowmeter and blood pressure transducer were connected to a computerized data acquisition system (MacLab; Colorado Springs, CO, USA). Analysis of CBF responses began 30 min after the end of the surgery to allow blood gases to stabilize. Animals with mean arterial blood pressure under 60 mmHg and/or blood gases outside the normal range (pH: 7.35–7.40; pCO₂: 33–45; and pO₂: 120–140) were eliminated from the study. CBF responses to neuronal activity were evaluated during whiskers stimulations. Three whiskers stimulations sessions of one minute were performed on the contralateral side of the CBF measurement. Three minutes of resting periods were left between each stimulation. CBF values were acquired with the LabChart6 Pro software (v6.1.3, AD Instruments, Colorado Springs, CO, USA). The percentage increase in CBF represents the peak CBF response relative to the resting CBF peak values during the 20 s before stimulations.