Investigative Ophthalmology & Visual Science, Vol. 33, No. 13, December 1992
Copyright © Association for Research in Vision and Ophthalmology
Accurate and Precise Measurement of Blood-Retinal
Barrier Breakdown Using Dynamic Gd-DTPA MRI
Bruce A. Berkowitz,* Paul 5. Tofts, f Harsha A. 5en4 Noburo Ando4 and Eugene de Juan, Jr^ :
Dynamic T,-weighted magnetic resonance imaging (MRI) after the injection of Gd-DTPA is a promis-
ing method for investigating breakdown of the blood-retinal barrier (BRB). Previously, the authors
demonstrated that in a T,-weighted image, the initial rate of change in the vitreous water MRI signal as
gadolinium diethylenetriaminepentaacetic acid (Gd-DTPA) enters the vitreous space strongly corre-
lated with the extent of BRB breakdown. Here, a practical approach to measuring a more relevant
physiologic parameter is presented: the permeability surface area product (PS). The theory is a develop-
ment of earlier work used in investigating the breakdown of the blood-brain barrier. The accuracy and
precision of this approach was investigated in rabbits pretreated with sodium iodate (30 mg/kg intrave-
nously). The MRI-derived PS normalized to the area of leaky retina (5.65 ± 0.25 X 10~4 cm/min, mean
± standard error of the mean; n = 6) was compared to a similarly normalized PS calculated using a
classical physiologic method (4.12 ± 0.73 X 10~4 cm/min; n = 6). Good agreement between the two
methods was found (P = 0.09). This result demonstrates that the MRI-derived PS is an accurate and
precise measure of BRB breakdown under these conditions. The mathematical model of Gd-DTPA
distribution in vivo also is validated. Based on these results, several potential sources of error are
discussed, including the effect of back-flow of Gd-DTPA from the vitreous space to the plasma, the
underlying vascular patency, and MRI slice selection. Invest Ophthalmol Vis Sci 33:3500-3506,1992
Recently, we demonstrated that dynamic, T,-
weighted magnetic resonance imaging (MRI) after in-
jection of the magnetic contrast agent gadolinium
diethylenetriaminepentaacetic acid (Gd-DTPA) is a
promising method for assessing breakdown of the
blood-retinal barrier (BRB) in the rabbit eye in vivo.1
When the BRB is disrupted, Gd-DTPA enters the vit-
reous space and increases the surrounding water pro-
ton relaxation rate (eg, [T,]"1) in direct proportion to
the Gd-DTPA concentration. As previously reported,
the rate of change in the vitreous water proton signal
after injection correlates with the degree of BRB
breakdown and relates to the permeability surface
area product (PS) of the lesion.1
A method for measuring PS from the initial part of
the enhancement curve recently was described.2 The
From the *National Institute of Environmental Health Sciences,
Research Triangle Park, North Carolina, *Duke University Eye
Center, Durham, and the flnstitute of Neurology, The National
Hospital, Queen Square, London, UK.
This project was partially supported by NIH research grant R01
EY07576 and the Juvenile Diabetes Foundation (EdJ). Bruce A.
Berkowitz is the recipient of a Research to Prevent Blindness Career
Development Award. H. A. Sen is a Heed Foundation Fellow.
Submitted for publication: April 10, 1992; accepted June 29,
1992.
Reprint requests: Bruce A. Berkowitz, Department of Ophthal-
mology, University of Texas Southwestern Medical Center, Dallas,
TX 75235-8592.
enhancement curve results from plotting the signal in
a T,-weighted sequence versus time after injection of
Gd-DTPA. The theory is a development of earlier
work used in investigating the breakdown of the
blood-brain barrier.3 Application of this approach
was performed on simulated MRI data of cerebral
multiple sclerosis and retinal lesions.2 However, di-
rect comparison between the MRI method and tradi-
tional physiologic methods of measuring PS have not
been performed. In the present study, after sodium
iodate breakdown of the outer BRB in the rabbit eye
in vivo, we first demonstrate the application of the
"Simplified Early Enhancement" method of Tofts
and Berkowitz2 to measure PS of the retina. Second,
we investigate the accuracy (systematic error) and
precision (reproducibility) of the PS derived from the
MRI data by comparing it to the PS obtained, on the
same eyes, using a classical physiologic method.
Materials and Methods
Animal Preparation
The 2-3 kg male or female mini-lop rabbits used in
this study were treated in accordance with Institu-
tional guidelines and the ARVO Resolution on the
Use of Animals in Research. Twenty four hours be-
fore the experiment, conscious animals were re-
strained in a rabbit box and given a bolus of 30 mg/kg
sodium iodate via the marginal ear vein. On the day of
3500

No. 13 QUANTITATIVE MEASUREMENT OF BLOOD-RETINAL BARRIER BREAKDOWN / Berkowirz er ol 3501
the experiment, the animals were anesthetized with a
mixture of ketamine HC1 (35 mg/kg) and xylazine
HC1 (5 mg/kg) intramuscularly. Anesthesia was
maintained by intravenous infusion of ketamine (20-
40 mg/kg/hr) and xylazine (1 mg/kg/hr) via the auric-
ular vein.4 The heart rate and blood pressure were
continually monitored from a femoral arterial cath-
eter, as previously described.1 The animal was artifi-
cially ventilated via an endotracheal tube.5 Blood gas
monitoring (micro 13; Instrumentation Laborator-
ies, Lexington, MA) was performed periodically to
ensure proper status of the animal. The rectal tempera-
ture of the animal was maintained at 37-38° C via a
circulating water blanket connected to a constant tem-
perature bath. The animal was gently placed in a
home-built nonmagnetic cradle and secured. At the
end of the experiments, the animals were killed by
KC1 injection intravenously.
Magnetic Resonance Imaging Procedure
All experiments were performed on a 4.7 T GE CSI
horizontal bore system (General Electric, Freemont,
CA) using a whole head, on-edge, split capacitance
Helmholtz coil (diameter 9 cm) tuned to 200 MHz.1
The animal was positioned so that a single coronal
slice went through the center of both eyes and was
oriented perpendicular to the myelin wing. Data were
collected using a standard spin echo sequence with TR
= 450 ms and TE = 28 ms, and a 3 mm slice thickness.
The field of view varied between 60 X 60 mm and 85
X 85 mm, depending on the head size. Images were
obtained with 128 phase encode steps, 256 complex
data points, and two acquisitions per phase encode
step. Each image took approximately 3 min to ac-
quire. The experimental protocol consisted of collect-
ing a control image before the introduction of the
contrast agent. Equal amounts of Gd-DTPA (0.5
mmol/1/kg; Magnevist; Berlex Labs, Wayne, NJ) and
saline then were injected over a 3 min period into the
ear vein. Setting the center of the injection as t = 0,
images were collected such that the zero phase encode
gradient came at t = 3.0,6.5, 11.5,16.5, and 21.5 min
after injection.
To determine the vitreous T, in the absence of Gd-
DTPA (T10, vide infra) a progressive saturation T, ex-
periment was performed. In a control rabbit, images
were obtained using parameters similar to those al-
ready mentioned, but with TR = 450 ms, 900 ms, 2
sec, 6 sec, and 10 sec (in random order). At each TR,
the signal intensity from both eyes were averaged to-
gether and this average value was fit to a standard T,
equation. The vitreous Tl0 value was 4.05 sec. In con-
trast, the vitreous T, value in vitro was 2.69 ± 0.02 sec
(mean ± standard error of the mean; n = 4). This
difference may have been due to the in vitro versus in
vivo temperature difference (25°C versus 37°C).6
Image analysis was performed on a Macintosh FX
II computer using the program Image (W. Rashban,
NIH, version 1.41) and consisted of defining a region
of interest (ROI) in one image, obtaining a mean sig-
nal intensity over that ROI, and applying that same
ROI to the other images in the set. In each eye, the
ROI for the vitreous was hand-drawn, conforming to
the entire retinal surface and including the complete
vitreous space. An external vial (1 cm inner diameter)
containing Gd-DTPA-doped water was included in
each image and provided a signal intensity standard
and the spatial calibration (number of pixels/cm)
needed for determining the area of the ROI and
length of the leaky portion of retina. The ROI signal
intensities normalized to the standard then were used
in the calculations. To minimize the contribution of
random error, PS at times 11.5, 16.5, and 21.5 min
post-injection were averaged together in each animal.
At these times, the enhancement was greater than
30%2 in all animals. Statistical analysis was performed
using a paired Student's t-test with P < 0.05 signifying
statistical significance.
To establish the Gd-DTPA concentration plasma
time course, blood samples (1 ml) were obtained in a
separate experiment in heparinized syringes during
the control period, immediately after the Gd-DTPA
injection, and 6.5, 11.5, 21.5, 31.5, 41.5, and 61.5
min post-injection. These samples were stored in ice.
At the end of the MRI experiments, the samples were
centrifuged and the plasma fraction was obtained for
NMR analysis. A simple inversion recovery T, exper-
iment was performed on the water signal of the
plasma fraction at 25°C. From the T, value, the
amount and thus concentration of Gd-DTPA was de-
termined from a calibration curve obtained at 25°C
in a separate phantom study using plasma from con-
trol animals.
The vitreous Gd-DTPA concentration at 60 min
post injection also was measured. Each animal exam-
ined by MRI was killed with KC1 intravenously. The
eyes were immediately enucleated and frozen by im-
mersion in liquid nitrogen. Differential tissue thawing
allowed the sclera, aqueous humor, iris, and lens to be
easily separated from the frozen vitreous. The vitre-
ous sample then was weighed to determine its vol-
ume, assuming a density of 1 gm/ml. After it reached
room temperature, the water proton T, was measured
for each vitreous sample (vide supra) and the Gd-
DTPA concentration was determined from a calibra-
tion curve previously obtained in a separate phantom
study at room temperature using vitreous from ani-
mals that had not received Gd-DTPA. The slope of
the calibration curve (Gd-DTPA concentration ver-