SUPPLEMENTARY INFORMATION
Supplementary Figure 1: MRI of Gadolinium (Gd3+)-DTPA-injected bovine eyes
shows flux of solutes into the lens. (A) Uninjected control bovine eye. The MRI signal
from the lens’ outer cortex appears uniform in all regions of the lens. The lens inner
cortex and nucleus appear relatively darker than the outer cortex due to their reduced
water content. Arrowheads demarcate the bow region (B) from the anterior epithelium
and lens cortex (A) and the posterior lens cortex (P). (B) Bovine eye injected with Gd3+DTPA. In the lens, the anterior outer cortex and the bow region of the lens display
stronger signal intensities than the posterior cortex of the corresponding control lens.
Further note the strong MRI signals in the vitreous body adjacent to the bow region. The
slice thickness is 500 µm. (C) Block charts showing the average signal intensities in the
anterior lens cortices, the bow regions and posterior lens cortices of the control and
three Gd3+-DTPA-injected eyes, respectively. These charts allow a direct comparison
between the signal intensities of these three regions in the different lenses. Note the
marked increase in signal intensity in the anterior cortices and the bow regions from
uninjected to injected eyes. (D) Pie charts showing the average signal intensities in the
anterior lens sector (top left sector; ant), the bow regions (top right sector; bow) and
posterior lens sector (bottom sector; post) of the control (1) and three Gd 3+-DTPAinjected eyes (2-4), respectively. A direct comparison between different regions of
control and injected lenses show that the posterior cortices in all injected eyes have
significantly less signal than the anterior cortices and the bow sectors.
It should be noted that these experiments only represent a limited and
preliminary study of the transfer of Gd3+-DTPA from the aqueous humour into the lens.
Clearly, more detailed experiments are needed before any hypotheses can be
confidently based on these observations. Such experiments should, for example, involve
different time-courses of incubation with Gd3+-DTPA, e.g. longer incubation times to test
whether even very slow diffusion occurs, the use of additional MRI markers as well as
experiments aimed at (i) determining whether the marker(s) remain extracellular and (ii)
confirming that the depth of penetration observed by MRI is truly greater than what could
be explained by simple diffusion.
SUPPLEMENTARY MATERIALS AND METHODS
6-Carboxyfluorescein Dye-loading Experiments
Adult rat eyes were dissected as previously described [1]. Lenses were placed in
tissue culture medium (Dulbecco's Modified Eagle's Medium (DMEM) with added 10%
v/v fetal calf serum, 1:100 Antibiotic Antimycotic Solution (0.1 g/l streptomycin, 200 U/l
penicillin and 0.25 mg/l amphotericine) and 2 mM L-glutamate; all Sigma) containing 30
µM 6-carboxyfluorescein-diacetate (Sigma) and incubated at 37°C and 5% CO2 for 5 min.
The lenses were subsequently washed three times in DMEM over a 30 min. period to
wash out non-decarboxylated fluorescein. Washed lenses were plunge frozen in liquid
nitrogen and sectioned (thickness: 30-40 µm) in a Reichert-Jung cryocut 1800 cryostat
(Leica, Vienna, Austria) at -20°C. The lens cryosections were fixed in a solution of 4%
(w/v) paraformaldehyde in PBS (Sigma) for 15 min, washed twice with PBS, incubated
with PBS containing propidium iodide (Sigma) at a final concentration of 1 µg/ml to stain
the nuclei and mounted for confocal microscopy. Fluorescence images were collected
using either a Zeiss LSM 410 unit (Carl Zeiss Jena GmbH, Jena, Germany) or a Biorad
MRC 600 LSM (Biorad Laboratories, Ltd., Hemel Hempstead, Herts) confocal laser
scanning microscope. Fluorescein was excited with the 488-nm line of a krypton-argon
laser and detected using a 515-565-nm band pass emission filter. Propidium iodide was
excited with the 568-nm laser line and detected with a 590-nm long pass emission filter.
All confocal images presented in this study are single optical sections. Images were
subsequently assembled into figures and labelled using the Adobe Photoshop CS
software (Adobe Systems Inc., San Jose, CA, USA).
MRI of Gadolinium-DTPA-injected Eyes
100 and 200 µl of DTPA-complexed gadolinium ions (non-ionic Omniscan
Gadodiamide; Nycomed Imaging AS, Oslo, Norway) were injected into the anterior
chamber of 1 hr. post-mortem adult bovine eyes (approximately corresponding to 1:25
and 1:12.5 dilutions of Gd-DTPA in aqueous humour, respectively). Trivalent Gadolinium
(Gd3+) is a paramagnetic element, which acts as an MRI contrast agent by substantially
decreasing T1 relaxation times in its surroundings. In contrast to the free ion, DTPAchelated Gd3+ is non-toxic and is routinely used in medical MRI applications, indicating
that it will not have adverse effects on lens physiology. Injected eyes were incubated in
PBS buffer at 37°C for 7-8 h prior to imaging in a Bruker AMX NMR spectrometer
operating at 300.13 MHz for protons, using a 65 mm internal diameter super-wide bore
imaging probe with a birdcage coil. Imaging was performed over a period of one hour for
individual optical sections through the eye. All images are cross-sectional spin echo
images through the eye’s visual axis with a pixel size of (195 µm) 2 and a slice thickness
of 100 or 500 µm. Images were subsequently assembled into figures and labelled using
the Adobe Photoshop CS software (Adobe Systems Inc., San Jose, CA, USA). Signal
intensities in the different lens regions were measured and averaged with the
MetaMorph 6.3 imaging software package (Universal Imaging/Visitron). While we cannot
exclude that the incubation of the eyes following the injection of Gd-DTPA resulted in
changes in the lens’ physiology that may have affected the diffusion of the MRI tracer
into the lens, it has recently been shown that the lenses in isolated sheep eyes retained
a normal water content for up to 30 h following eye removal from the orbit [82, 2].
Furthermore, all lenses examined in this study were inspected for changes to lens clarity
following the experiment.
Detailed descriptions of the histological and electron microscopic techniques
used can be found in the papers published by the authors included in the list of
references.
References
1.
Dahm, R., Procter, J. E., Ireland, M. E., Lo, W. K., Mogensen, M. M., Quinlan, R. A. & Prescott, A.
R. 2007 Reorganization of centrosomal marker proteins coincides with epithelial cell differentiation
in the vertebrate lens. Exp. Eye. Res. 85, 696-713. (doi: 10.1016/j.exer.2007.07.022)
2.
Augusteyn, R. C. & Cake, M. A. 2005 Post-mortem water uptake by sheep lenses left in situ. Mol.
Vis. 11, 749-751