To correct for any movement (e.g., subtle shifting of the animals’ position occurred during the experiment due to settling) in the slice plane, a warp affine image coregistration was performed on each animal by using software written in-house. We insured that the selected slice was the same one used throughout the series by carefully checking for differences in the size of lens and optic nerve in each image. In addition, because the slice thickness (1 mm) is relatively large compared with the diameter of the eye (∼6 mm), partial volumes will remain similar if the eye subtly moves out of the imaging plane, and so the data analysis results are not expected to be substantially affected. After coregistration, the MRI data were transferred to a computer (Power Mac G4; Apple Computer, Cupertino, CA) and analyzed with NIH Image (a freeware program available 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. Signal intensity changes during carbogen breathing were calculated and converted to ΔP
o 2, on a pixel-by-pixel basis, as follows.
8 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 (with
t 1 representing the first 2 minutes and
t 2 representing the second 2 minutes of carbogen), and
S 0 is the control signal intensity (measured from the average of the three images obtained during room air breathing) at the same pixel spatial location.
E values were converted into ΔP
o 2 using theory that has been validated in the rat
10 :
\[{\Delta}\mathrm{P\mbox{\textsc{o}}}_{2}{=}E/(R_{1}\ {\ast}\ T_{k}),\]
where
R 1 is the oxygen relaxivity (s
−1 · mm Hg
−1), and
T k is
T r · exp(−
T r/
T 10),
T r is the repetition time, and
T 10 is
T 1 in the absence of oxygen. Using a
T r of 1 s, and assuming a vitreous
T 10 of 4 s,
T k = 3.52. This
T 10 is based on our previous measurement of the proton spin-lattice relaxation time in the rabbit vitreous (4 s), reported values in human vitreous (3.3 s), and those in cerebral spinal fluid (4.3 s), which has a high water content similar to that of vitreous.
8 11 12 13 An
R 1 of 2 × 10
−4 s
−1 · mm Hg
−1 was used. This
R 1 was previously measured in a saline phantom, which is assumed to be a reasonable model of vitreous (98% water).
8 A similar
R 1 was found for plasma suggesting that relatively low protein levels do not substantially contribute to the oxygen relaxivity.
14 Note that an
E of 0.01 (i.e., a 1% signal intensity change) corresponds to a ΔP
o 2 of 14 mm Hg. There did not appear to be any significant changes in vitreous
T 10 or
R 1 in the animals studied (data not shown).