To correct for possible subtle shifting of the animals’ position during the experiment (usually for reasons unrelated to the physiology of the animal, such as settling on the gauze packing) a warp affine image coregistration was performed on each animal with software written in-house. After coregistration, the MRI data were transferred to a computer (Power Mac G4; Apple Computer, Cupertino, CA) and analyzed with the software program Image (available by ftp at zippy.nimh.nih.gov/ or at http://rsb.info.nih.gov/nih-image; developed by Wayne Rasband, National Institutes of Health, Bethesda, MD). Images obtained during room air breathing were averaged to improve the signal-to-noise ratio. All pixel signal intensities in the average-room-air image and the 2-minute carbogen image were then normalized to the external standard intensity. Signal intensity changes during carbogen breathing were calculated and converted to ΔP
o 2 values, on a pixel-by-pixel basis, as follows.
13 For each pixel, the fractional signal enhancement (
E) was calculated
\[E\ {=}\ (S(t)\ {-}\ S_{0})/S_{0}\]
where
S(
t) is the pixel signal intensity at time
t after starting the gas inhalation, and
S 0 is the control signal intensity (measured from the average of the three control images) at the same pixel spatial location.
E values were converted into ΔP
o 2 according to a previously established theory
13 \[{\Delta}\mathrm{P\mbox{\textsc{o}}}_{2}\ {=}\ E/(R_{1}^{{\ast}}T_{\mathrm{k}})\]
where
R 1 is the oxygen relaxivity (second
−1 · mm Hg
−1), and
\(\mathit{T}_{k}\ {=}\ \mathit{T}_{r}\ {\cdot}\ exp^{({-}\mathit{T}_{r}/\mathit{T}_{10})}\) , where
T r is the repetition time, and
T 10 is the
T 1 in the absence of oxygen. Using a
T r of 1 second and assuming a vitreous
T 10 of 4 seconds,
T k = 3.52. This
T 10 value is based on our previous measurement of the proton spin–lattice relaxation time in the rabbit vitreous (4 seconds) and reported values in human vitreous (3.3 seconds) and in cerebral spinal fluid (4.3 seconds), which has a high water content similar to that of vitreous.
13 25 An
R 1 of 2 × 10
−4 sec
−1 · mm Hg
−1 was used.
13 This
R 1 was previously measured in a saline phantom, which is assumed to be a reasonable model of vitreous (98% water).
13 A similar
R 1 value was found for plasma, suggesting that relatively low protein levels do not substantially contribute to oxygen relaxivity.
26 Note that an
E of 0.01 (i.e., a 1% signal intensity change) corresponds to a ΔP
o 2 of 14 mm Hg.