The general mouse preparation for high-resolution MRI is well established in our laboratory.
5 All animals were maintained in darkness for at least 16 hours before and during the dark phase of the MRI examination. In some mouse groups, MnCl
2 was administered, under dim red light or in darkness, as an intraperitoneal injection (66 mg MnCl
2·4H
2O/kg) on the right side of awake mice.
3,41 After this injection, mice were maintained in the dark for another 3.5 to 4 hours. High-resolution anatomic and ADC data were acquired on a 7 T system (Bruker ClinScan; Billerica, MA, USA) using a receive-only surface coil (1.0 cm diameter) centered on the left eye. The end of a fiber optic bundle was attached to a light source (Mark II Light Source; Prescott's, Inc., Monument, CO, USA) placed caudal to the eye, projecting at a white screen ~1 cm from the eye, similar to that previously described.
19 We exposed the eye to 0 (i.e., dark) or ~500 lux (confirmed outside the magnet using a Traceable Dual-Range Light Meter [Control Company, Friendswood, TX, USA]) placed against a 1-cm-diameter aperture; measured this way, room lighting is ~300 lux). Aside from the fiber optic light source, all lights in the MRI room were turned off. In all groups, immediately before the MRI experiment, animals were anesthetized with urethane (36% solution intraperitoneally; 0.083 mL/20 g animal weight, prepared fresh daily; Sigma-Aldrich Corp., St. Louis, MO, USA) and treated topically with 1% atropine to ensure dilation of the iris during light exposure, followed by 2% lidocaine to reduce eye motion. Anatomic and ADC (parallel to the optic nerve, the most sensitive direction for detecting changes at the location of the SRS
19) MRI data sets were collected, first in the dark and then again 15 minutes after turning on the light; since each ADC data set takes 10 minutes to collect, we refer to the midpoint in the ADC collection as 20 minutes of light exposure. The subset of nondiabetic wt mice in which an ADC data set was collected immediately after turning on the light was called the 5 minutes of light exposure time point group. Anatomic images were acquired using a spin echo sequence (slice thickness 600 μm, TR 1000 ms, TE 11 ms, matrix size 192 × 320, field of view 8 × 8 mm
2, NA 2, axial resolution for central retina 25 μm); images sensitized to water diffusion were collected (TR 1000 ms, slice thickness 600 μm, TE 33 ms, matrix size 174 × 288, field of view 8 × 8 mm
2, axial resolution for central retina 27.8 μm;
b = 0, 100, 250, 500, 600, 750, 990 s/mm
2 [collected in pseudorandom order, NA 1 per
b value]), registered to the anatomic image, and analyzed (using in-house code) to generate ADC profiles from the central retina. The present resolution in the central retina is sufficient for extracting meaningful layer-specific anatomic and functional data, as previously discussed.
42 For example, given the present whole-retinal thicknesses of ~238 μm (the average thickness across controls in the
Table) and the pixel size (~26 μm [mean of 25 and 27.8 μm]), each pixel axially spanned approximately 11% thickness or ~9 μm. We also note that the uncertainty in a pixel's thickness can be estimated to be ~½ pixel thick. The data support this estimate because converting all of the SEM back to SD from the
Table and averaging gives a variance of ~13.5 μm, which is in reasonable agreement with the ½ pixel values of 12.5 to 13.9 μm (from anatomic to ADC images). The data also support our ability to distinguish changes in central retinal thickness on the anatomic images and significant ADC changes at 88% to 100% depth. In all cases, anesthetized animals were humanely euthanized by cervical dislocation followed by bilateral pneumothorax for assurance of death per our DLAR-approved protocol.