Non-invasive brain ultrasound with transcranial echo-color doppler (IE 33, Philips Medical System, the Netherlands) is performed in three steps to 1) evaluate the global cerebral blood volume, 2) to estimate the presence or absence of cerebral autoregulation, and 3) to qualitatively evaluate the cerebral perfusion and microcirculation by enhanced microbubbles contrast injection (CEUS). Brain ultrasound is performed in the first 48 hours after hemodynamic stabilization (MAP > 65mm Hg, normotherm) and respiratory parameters stabilization (PaCO2 between 35-45mmHg and the PaO2/FiO2 > 200) after ICU admission. The second and third examination is performed in the next 48-72 hours before sedation withdrawal to limit the effect of change of cerebral hemodynamics.

Before performing brain ultrasound, echocardiography (IE 33, Philips medical System, the Netherlands) is performed to evaluate left ventricular ejection fraction and cardiac output (L/min).

First, the global cerebral blood volume (L/min) is evaluated as the sum of flow volumes of the internal carotid (ICA) and vertebral arteries (VA) extracranial arteries of both sides. The internal carotid artery (ICA) is examined with a 7-MHz linear array transducer with the head of the patient slightly titled upward, in midline position. The site of measurement is approximately 1.5cm below the carotid bulb in the in the common carotid artery (CCA) during expansion and 1.5cm away from the bifurcation in ICA. In the presence of atheromatous calcifications plaques, ICA doppler measurement is performed outside and before the plaques. The B-mode bidimensional is magnified to achieve a higher resolution and details. The internal diameter of the vessel is measured at the exact site of the pulse doppler velocity measurement ample volume, between both endothelial layers, perpendicular of the course of the vessel. The diameter of the vertebral artery is examined and magnified in B-mode. The transducer is positioned along the CCA, shiftily laterally and angled until the intertransverse segment of the VA is seen and the doppler velocity is measured at the C4-C5 transverse area along the common carotid artery exactly at the same place of diameter measurement. The following measurements of flow velocities are taken in each artery: Peak systolic and end-diastolic velocity, time-averaged velocity (TAV), Pulsatility Index (PI). Flow volume (Q) of each artery is determined as Q = TAV x Area ((diameter of the artery /2) x PI).

Transcranial echo-color doppler is performed via temporal windows and the Pulsatility Index (PI) and the mean flow velocities (cm/sec) are measured of MCA, at both sides are recorded. Cerebrovascular resistance index as defined as the ration MAP/Mean Flow velocity of MCA (mmHg/cm per second).

Second, after measuring the global cerebral blood volume, the presence or absence of cerebral autoregulation (CA) is tested with the Transient hyperemic response (THR) by measuring the velocity of the media cerebral artery (MCA) the following an ipsilateral common carotid compression during 8 seconds. THR is defined as the F3/F1 ratio F1 as the MCA velocity before compression and F3 is the second MCA velocity after compression test). THR test is valid when onset of compression results a sudden and maximal decrease in MCA blood velocity and remains stable during compression.

Third, after testing the THR, the brain regional microcirculation is evaluated by the microbubbles contrast injection SONOVUE (Italy) following the European guidelines recommendation for contrast microbubbles enhanced ultrasound. The brain parenchyma is insonated via the temporal bone windows at the depth of 10cm with the ultrasound S5 multifrequency transducer 2-5 MHz probe. After optimizing the acoustic bone window, SONOVUE is injected intravenously as a bolus 2.4ml followed by 10ml saline flushed. The contralateral brain is evaluated 5 minutes after the first injection of SONOVUE to allow a complete evacuation of contrast microbubbles.

All real-time CEUS images were stored digitally on the hard disk as DICOM (Digital Image Communications in Medicine) images. Offline analysis used the QLAB 10 quantification software (Philips Medical System, the Netherlands) to convert brain perfusion images into time-intensity curves (TIC) corresponding to the five different regions of interest (ROI) of brain parenchyma: anterior and posterior thalamus, lentiform nucleus, parieto-temporal and posterior white matter. Four variables were extracted from these TIC curves to qualitatively evaluate the brain microcirculation: peak intensity in dB (PI), time to peak intensity in seconds (TTP), mean transit time in seconds (MTT), and area under the curve in dB/seconds (AUC) .