The generation of dOHb as a contrast agent has seldom been exploited for hemodynamic studies due to the difficulty in precisely targeting arterial PO2 (PaO2) by the inspired PO2. This difficulty is compounded by the requirement of simultaneously and strictly maintaining a constant arterial PCO2 (PaCO2) in order not to disturb the cerebral blood flow. Once the means of achieving these targeted lung gas transitions is solved, the physiology of gas-blood equilibration is very advantageous to the control of contrast formation. Inspired gas instantaneously distributes widely throughout the lungs, roughly matching pulmonary blood flow. The very large diffusion interface (50-75 m 2 , about half the size of a tennis court) then results in a near instantaneous equilibration of the lung PO2 with that of mixed venous blood, and hence the corresponding [dOHb] which is conducted directly to the systemic arterial circulation.
For over 20 years rapid precisely targeted lung and arterial blood PCO2 have been induced by the method of sequential gas delivery (SGD) 7 care. Despite the small variations in protocols, the investigators found the generated data remarkably consistent, and of sufficiently high contrast to noise ratio (CNR) to merit an early alert to the imaging community. In this communication, we are able to report: 1) the successful implementation of an abrupt 10 s reduction in [dOHb] and its BOLD response; 2) the BOLD contrast-to-noise ratio (CNR) resulting from the change in [dOHb] ; 3) the degree of [dOHb] bolus dispersion upon arrival in arteries of the brain constituting an effective arterial input function; 4) calculation of standard blood flow parameters that are consistent with those in previous publications.
As these methods for inducing [dOHb] changes are totally unprecedented, we were obliged to perform initial exploratory studies to develop the study protocols prior to the IRB application. IRB considered these studies developmental nature and that all subjects were investigators and thus well informed, and did not require full IRB review.
All scanned investigators were in good health without contraindications to MRI environment and brief episodes of mild hypoxia. Each provided signed consent for the studies.
An investigational computerized gas blender, the RespirAct™ RA-MR (Thornhill Medical, Toronto, Canada), was used to prospectively target lung PO2 while maintaining normocapnia independently of ventilation and target lung PO2. 8, 9 End-tidal PO2 (PETO2) was lowered to a baseline of about 55 mmHg (hypoxia baseline) while maintaining normocapnia in order to minimize the changes in arterial PCO2 (and hence its effect on cerebral blood flow). Hypoxia was maintained for 60 to 90s followed by a gas challenge with the PETO2 target set to 100 mmHg for 10s to transiently saturate hemoglobin with O2 before returning to baseline. This normoxic challenge and return to hypoxic baseline was then repeated 3 to 4 times during each trial ( Figure 1 ). The recorded end-tidal PETO2 was converted to SaO2 using the Hill equation normal sea level physiologic parameters 6 : pre-processing steps for the whole brain dataset with TR of 2000ms included slice time correction, volume re-registration, and fourth degree polynomial detrending. SaO2 was resampled and interpolated to TR intervals and time-aligned to the whole brain average BOLD signal.
The baseline BOLD signal (S 0 ) was then defined as the mean of the BOLD signal (S t ) over five time-frames including the duration of the baseline before the first bolus, 10 sec before the second, third and fourth boluses, and 10 sec before the end of the protocol.
The scaled BOLD signal (S c,t) could then be calculated as
A time delay (TD) map was calculated using cross-correlation between 1,3 and multiple Standard tracer kinetic modeling was used to calculate MTT and CBF as per:
Subscript t indicates that the variable is a function of time.
SaO2 was used as the arterial input function (AIFt) and was scaled to have an AUC equal AUCv.
3 = +3/?@@ was used for the residue function. It is equal to 1 at time 0 and set to 0 at time equal to 5 x MTT. CBF and MTT can then be determined using least square fitting procedure. MTT was bound between 0 and 7s.
The T1-weighted images were segmented into gray matter, white matter and cerebrospinal fluid using spm8 software package 11 . The probability density maps obtained were thresholded at 0.8 to generate a mask for gray matter (GM) and white matter (WM). One layer of peripheral voxels was eroded from the white matter mask to minimize partial volume effects.
Gray and white matter mask were used to calculate average values of BOLD signal, CNR, TD, MTT, CBV and CBF. A paired t-test was also computed to assess the change in CNR for each set of merged boluses.
: Figure 1 illustrates the extent of dispersion inclusive of that in the lung, confluence in the pulmonary vein, wash-in and wash-out of the left ventricle, and any mixing in the aorta and extracranial arteries. There is also remarkable consistency in the successive gas challenges and resulting BOLD changes with little drift (Figure 1 b) . 12 
We report remarkable findings in the course of protocol development for studies in using As expected, the rise in the arterial signal precedes the rise in the venous signal. The larger rise in the venous BOLD signal is consistent with a lower baseline of the unscaled BOLD signal. The reason for this is that large veins/sinuses are bigger than large arteries, and more likely to contain an imaging voxel with 100% blood that has high [dOHb] and therefore low signal than voxels over arteries where voxels that include both arterial blood and adjacent tissue that has only 4% blood volume. Therefore when scaling the image, dividing by the lower baseline in veins yields a higher percent change in BOLD signal over veins.
Induction of hyperoxia has been used by others to induce BOLD signal changes to enable measurement of CBF, CBV and MTT 17 . The signal characteristics with hyperoxia differ from intravascular bolus contrast or from abrupt inhalations as in our method. Finally, in terms of safety to exposure to brief periods of hypoxia, there is no evidence that it is harmful. For example, in La Paz, the capital city of Bolivia at 4000 m elevation, most of the population, as well as visitors have PO2 in the range of 50-60 mmHg, which may be associated with some limitation in exercise tolerance, but not serious illness. For further perspective, most of the human population undergoes multiple (5-25) episodes of apnea (cessation of breathing) during sleep. PaO2 has frequently been observed to fall to values 30-40 mmHg 6 . Thus, the PaO2 of 50-55 mmHg is well tolerated by humans and frequently encountered as part of day-to-day living. In our study, the low PaO2 at baseline was virtually imperceptible, causing no distress to any of the 5 subjects.
In this study, hypoxia was established as a baseline, and a normoxic lung changes with decreasing [dOHb] used as the stimulus. The same imaging principles would apply in the reverse situation with a normoxic baseline and hypoxic gas challenges with increasing