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Experimental Eye Research 87 (2008) 334–341
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Experimental Eye Research
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GD-DTPA enhanced MRI of ocular transport in a rat model of chronic glaucoma
Kevin C. Chan a, b, Qing-ling Fu c, d, Hua Guo a, b, Kwok-fai So c, d, Ed X. Wu a, b, *
a
Laboratory of Biomedical Imaging and Signal Processing, The University of Hong Kong, Pokfulam, Hong Kong
Department of Electrical and Electronic Engineering, The University of Hong Kong, Pokfulam, Hong Kong
c
Department of Anatomy, The University of Hong Kong, Pokfulam, Hong Kong
d
The State Key Laboratory of Brain and Cognitive Sciences, The University of Hong Kong, Pokfulam, Hong Kong
b
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 29 April 2008
Accepted in revised form 23 June 2008
Available online 28 June 2008
Glaucoma is a neurodegenerative disease of the visual system characterized by the elevation of intraocular pressure. While this elevated pressure is related to an increased resistance to the outflow of
aqueous humor from the eye, their impacts to the etiology and pathogenesis of the disease are not fully
understood. This study aims to employ in vivo Gd-DTPA enhanced magnetic resonance imaging to
evaluate the ocular transport following an induction of ocular hypertension in a rat model of chronic
glaucoma. An experimental ocular hypertension model was induced in adult rats using an argon laser to
photocoagulate the episcleral and limbal veins on the surface of the eyeball. The enhancements of the
MRI signal intensity in the anterior chamber and vitreous body were measured as a function of time
following systemic administration of Gd-DTPA solution at 3 mmol/kg. Results showed a progressive T1weighted signal increase in the vitreous body of the glaucomatous eye but not the control eye. This
increase occurred earlier in the anterior vitreous body than the preretinal vitreous. Further, there was an
earlier Gd-DTPA transport into the anterior chamber in the majority of glaucomatous eyes. Our findings
revealed the leakage of Gd-DTPA at the aqueous–vitreous interface, which was likely resulted from increased permeability of blood–aqueous or aqueous–vitreous barrier. These may explain the sources of
changing biochemical compositions in the glaucomatous chamber components, which may implicate the
cascades of neurodegenerative processes in the retina and the optic nerve.
Ó 2008 Elsevier Ltd. All rights reserved.
Keywords:
chronic glaucoma
intraocular pressure
ocular transport
aqueous–vitreous interface
Gd-DTPA enhanced magnetic resonance
imaging
1. Introduction
Glaucoma is a neurodegenerative disease of the visual system
characterized by an increase in intraocular pressure (IOP). It is the
second major cause of blindness in the world (Quigley and Broman,
2006). While the level of IOP is governed by the dynamic balance
between aqueous humor formation and drainage (Morrison et al.,
1995), the elevated IOP in primary open-angle glaucoma (POAG) is
believed to arise due to an increased resistance to the outflow of
aqueous humor from the eye (Johnson, 2006). Nevertheless, due to
the lack of a non-invasive method to monitor the inner-depth of the
eye without critically affecting the dynamic behavior of the aqueous outflow system (Camelo et al., 2004; Carreras et al., 1997;
Kolodny et al., 1996; Manfre et al., 1997; Mermoud et al., 1996), the
aqueous outflow obstructions in glaucoma have not been well
understood (Kaufman, 1999).
Magnetic resonance imaging (MRI) allows inner-depth localization of dynamic ocular processes in vivo (Alikacem et al., 2000;
* Corresponding author. Laboratory of Biomedical Imaging and Signal Processing,
Department of Electrical & Electronic Engineering, The University of Hong Kong,
Hong Kong. Tel.: þ852 2819 9713; fax: þ852 2819 9711.
E-mail address: [email protected] (E.X. Wu).
0014-4835/$ – see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.exer.2008.06.015
Berkowitz et al., 2004; Bert et al., 2006; Chan et al., 2008; Freddo et al.,
2006; Kim et al., 2005, 2007, 2008; Kolodny et al., 1996, 2002; Li et al.,
2004a,b, 2008a,b; Manfre et al., 1997; Molokhia et al., 2007, 2008;
Patz et al., 2007; Trick et al., 2005; Wilson et al., 1995). Recent studies
using the arterial spin labeling (ASL) technique showed high basal
blood flows in the ciliary body and retina of rat eyes (Li et al., 2008b).
It was also shown that MRI can detect multiple anatomical retinal
layers, and that the retinal and choroidal vascular layers bounding the
retina can be detected using an exogenous MR contrast agent gadolinium-diethylenetriamine pentaacetic acid (Gd-DTPA) (Cheng et al.,
2006; Shen et al., 2006). In the ciliary body, the aqueous humor is
produced primarily by active transport across epithelial cells lining
the surface of the ciliary processes (Jacob and Civan, 1996; Raviola
and Raviola,1978), and is drained away from the venous plexus of the
limbus via a circumferential Schlemm’s canal into multiple radial
veins located within the episclera (Morrison et al., 1995). This conventional route of aqueous humor outflow (McMenamin and alShakarchi, 1989) taken by plasma-derived proteins has been traced
using contrast-enhanced MRI (Bert et al., 2006; Kolodny et al., 1996),
by demonstrating the entrance of Gd-DTPA from the bloodstream
into the ciliary body stroma, and its exit via the iris root (Kolodny
et al., 2002). This potentiated the study of changes in aqueous outflow
in the rat model of chronic glaucoma using Gd-DTPA enhanced MRI.
K.C. Chan et al. / Experimental Eye Research 87 (2008) 334–341
One of the most commonly used experimental glaucoma
models involves laser photocoagulation of the episcleral and limbal
veins, which mimicks the pathogenesis of human POAG (Barsotti
et al., 1992; Li et al., 2006b). It has been suggested that biochemical
changes in the chamber humor played an important role in the
pathogenesis of POAG and were largely determined by the permeability of the blood–ocular barrier (Kuryshova et al., 1998). This
might be due to dystrophic changes in ocular tissues in the course
of glaucoma progression, and accumulation of metabolites damaging the biological membranes in the chamber fluid, such as
vascular endothelial growth factor (VEGF) (Aiello et al., 1994; Hu
et al., 2002), endothelin-1 (ET-1) (Prasanna et al., 2005) and other
proteins (Lee et al., 1990). It was also suspected that ocular hypertension attributed to a compromise of the aqueous–vitreous barrier
integrity. The lens-zonule system comprising the posterior capsule,
the anterior hyaloid membrane and the crystalline lens forms
a diaphragm between the anterior chamber and vitreous body,
blocking large nonelectrolytes and negatively charged electrolytes
such as lipids, proteins, hyaluronic acid, and enzymes (De Groot
et al., 2003; Ozaki, 1984). Disruption of this barrier might lead to
a quick rise in anterior vitreous fluorescence by increased diffusion
from the anterior chamber (De Groot et al., 2003). It is known that
upon perfusion of the anterior chamber at elevated pressures, the
pressure in the anterior chamber was communicated to the lens
(Carreras et al., 1997) and the posterior displacement of the lens
stretched the zonular ligament (Van Buskirk, 1980). This pulled
back the detached portion of the ciliary body leading to the aperture of the ciliary cleft (Carreras et al., 1997). Our previous studies
using intravitreal injection of manganese chloride have also shown
an abnormally higher T1-weighted signal intensity in the vitreous
body of the glaucomatous eye 5 h after administration, which
appeared to result from the perturbation of the usual pattern of
Mn2þ clearance in the glaucomatous eyeball upon the blockade of
aqueous outflow (Chan et al., 2008).
Despite the relatively small molecular mass of Gd-DTPA
(938 Da), in normal intact eyes, the blood–retinal barrier and
aqueous–vitreous barrier are impermeable to Gd-DTPA (Berkowitz
et al., 2004; Bert et al., 2006; Kolodny et al., 1996; Trick et al., 2005;
Wilson et al., 1995). Gd-DTPA enhanced MRI has been used to examine the pathology that altered the permeability of blood–aqueous barrier (Freddo et al., 2006; Galluzzi et al., 2003; Kolodny et al.,
2002) and blood–retinal barrier (Berkowitz et al., 1991, 2004; Sato
et al., 1992; Trick et al., 2005; Wilson et al., 1995) in both human and
animal eyes. On the basis of the previous findings, this study aims to
employ high resolution Gd-DTPA enhanced MRI to understand the
ocular transport following an induction of ocular hypertension in
a rat model of chronic glaucoma. The enhancements of the MRI
signal intensity in the anterior chamber and vitreous body were
measured as a function of time following systemic administration
of Gd-DTPA. We hypothesized that systemic administration of GdDTPA into our glaucoma model would detect whether leakage
existed at the aqueous–vitreous interface giving rise to a progressive signal enhancement in the anterior vitreous body of the
glaucomatous eye. Further, we attempted to monitor the transport
of Gd-DTPA into the anterior chamber of our glaucoma model. Such
phenomena may help understand the sources of biochemicals
present in the chamber components of the glaucomatous eye participating in the cascades of neurodegenerative processes in the
retina and the optic nerve.
2. Methods
2.1. Animal preparation
Sprague-Dawley rats (250–280 g, N ¼ 11) were divided into two
groups and were reared in a temperature-controlled room
335
subjected to a 12 h light/dark cycle with standard chow and water
supply ad libitum. They were induced for ocular hypertension
unilaterally in the right eye by photocoagulation of three episcleral
veins and the limbal veins on the surface of the eyeball using an
argon laser to maintain a consistent IOP elevation by 1.6 times
above the normal level as demonstrated up to a maximum of 12week experimental period (Li et al., 2006a,b). This technique was
modified from the method by WoldeMussie et al. (2001) and has
been adopted in our laboratory for the study of retinal degeneration
and therapeutic effects on retinal functions (Chan et al., 2007, 2008;
Fu et al., 2008a,b; Li et al., 2006a,b). After each procedure, antibiotic
ointment was applied topically to the eye surface. Gd-DTPA enhanced MRI was performed at Day 5 (Group 1, n ¼ 5) and Week 8
(Group 2, n ¼ 6) after laser treatment to investigate the early and
late effects of glaucoma induction.
2.2. MRI protocols
All MRI measurements were acquired on a 7T MRI scanner with
a maximum gradient of 360 mT/m (70/16 PharmaScan, Bruker
Biospin GmbH, Germany) using a 72 mm birdcage transmit-only RF
coil with an actively decoupled receive-only quadrature surface
coil. The rat was placed onto a head holder comprising a tooth bar.
Under inhaled isoflurane anesthesia (3% induction and 1.5% maintenance), the animal was kept warm under circulating water at
37 C. A saline phantom was inserted beside the rat for signal intensity normalization during post-processing. Five days (Group 1)
and 8 weeks (Group 2) after laser treatment, Gd-DTPA (Magnevist)
was applied systemically by intraperitoneal injection at 3 mmol/kg,
and 2D fast spin-echo (FSE) T1-weighted imaging (T1WI) was applied before and 10–80 min after injection to track the movement
of the contrast material from the bloodstream into the tissues and
compartments of the eye. Throughout the experiments, the left eye
served as a control. Scout anatomical images were first acquired in
three orthogonal planes with multi-slice FSE T2-weighted imaging
(T2WI) so as to guide the subsequent high resolution images to
bisect the center of the eye and the optic nerve head in a reproducible manner. High resolution T1WIs were acquired using
a 2D FSE sequence with fat suppression, TR/TE ¼ 320/8.9 ms, echo
train length ¼ 4, NEX ¼ 26, slice thickness ¼ 1 mm, slice
gap ¼ 0.1 mm, no. of slices ¼ 4, FOV ¼ 3.2 3.2 cm3 and voxel resolution ¼ 82.3 82.3 mm3. Acquisition time was 10 min for each
time point. Between 0 and 10 min after injection, T1WIs were also
acquired in Group 1 with the same T1WI protocol above but with
NEX ¼ 5 and acquisition time ¼ 2 min for each time point to monitor the enhanced pattern immediately after injection.
2.3. Data analysis
In the 2D slice that centrally cut through the eyeball and optic
nerve head, regions of interest (ROIs) were drawn manually on the
whole anterior chamber, and on the vitreous body covering a distance of 3.5 mm on each side from the optic nerve head using
ImageJ v1.38 (Wayne Rasband, NIH, USA) as shown in Fig. 1. An
effort was made to maximize the number of pixels analyzed
without creating partial volume effects by avoiding the regions’
margins while keeping the voxel number in the ROIs constant for
each compartment. Each value was calibrated to the nearby
phantom containing saline solution to avoid the effect of any MRI
system sensitivity drift. A quantitative measure of the Gd-DTPA
entry was obtained from the enhancement in the MR signal intensity defined as E ¼ [S(t) So]/So, where S(t) is the calibrated
spin-echo signal intensity of the ROIs, and So is the T1-weighted
signal intensity of the same ROI before the MR tracer injection.
Differences between mean values of the ROIs on both sides were
compared using two-tailed paired t-tests, and the mean values
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K.C. Chan et al. / Experimental Eye Research 87 (2008) 334–341
Fig. 1. Illustration of the typical ROI definitions used to quantify the Gd-DTPA enhanced MRI measurements. The regions of interest (ROIs) include anterior chamber (AC) and
vitreous body (VB). The lens (Ln) and optic nerves (ON) could be observed as hypointense signals in both pre- (left) and post- (right) systemic Gd-DTPA injection images, while the
hyperintense ciliary processes could also be delineated between the posterior chamber (PC) and the vitreous body after systemic Gd-DTPA injection (right).
along the time course were compared using ANOVA. Results were
considered to be significantly different when p < 0.05.
The enhanced anterior chamber volume across all scanned slices, and the axial eye length in the centrally cut 2D slice were
computed and compared between contralateral eyes at the end
time point (80 mins post-administration of Gd-DTPA) of each experiment. The anterior chamber volume was the volume between
the anterior corneal surface to the anterior lens surface, and the
axial eye length was the distance from the anterior corneal surface
to the retinal pigment epithelium (Leydolt et al., 2007).
3. Results
Fig. 1 shows the typical ROI definitions used to quantify the GdDTPA enhanced MRI measurements, and Fig. 2 shows the serial
T1WIs of the glaucomatous and control eyes early after systemic
Gd-DTPA administration. Fig. 3 shows the mean percent signal
enhancement, E, as a function of the time for the anterior chamber
and vitreous body in the glaucomatous and control eyes. In the
control eye, there was an immediate enhancement of the ciliary
processes, iris and chorioretina in all scanned rats after Gd-DTPA
administration (Fig. 2). The anterior chamber enhanced substantially over the first 40–60 min after Gd-DTPA administration
(Fig. 3) (ANOVA, p < 0.01), while the vitreous and lens did not enhance at any time (ANOVA, p > 0.05). These patterns of T1weighted signal enhancements generally followed as previously
described (Alikacem et al., 2000; Bert et al., 2006; Kolodny et al.,
1996, 2002).
In the glaucomatous eye, however, the vitreous body enhanced
progressively from 10 min post-administration in both groups after
glaucoma induction (Figs. 3 and 4) (ANOVA, p < 0.01). Significant
difference was observed between the glaucomatous and the control
eyes in Group 2 (paired t-test, p < 0.05), and was marginally observed in Group 1 (paired t-test, p < 0.07 for the later 2 time points).
No apparent difference was observed between the two groups on
the signal changes in the glaucomatous eyes (unpaired t-test,
p > 0.05). Qualitative inspection of the MR images of both groups
suggested that Gd-DTPA leaked at the aqueous–vitreous interface
and diffused into the vitreous body from the anterior parts of the
glaucomatous eye in all rats, while no apparent signal increase was
observed in the same location in the control eyes (Fig. 4). Note that
in 3 out of the 5 rats in Group 1, the intensity in the anterior
chamber was greater for the glaucomatous eye than the control one
in the first 10 min after Gd-DTPA administration (Fig. 2). For Group
2, 4 out of 6 rats had their signal intensities higher in the glaucomatous anterior chamber than the control throughout the
experiment.
For the dimensions of the eye components, while no apparent
difference was found in the axial eye length between contralateral
eyes (data not shown), the anterior chamber volume was significantly increased in the glaucomatous eyes when compared to the
control eyes in both Group 1 (12.24 3.06 mm3 vs
Fig. 2. Serial T1-weighted images (T1WIs) of a rat at Day 5 (Group 1) after glaucoma induction. Images were acquired within the first 10 min following systemic Gd-DTPA administration. Note that in 3 out of the 5 rats in the group, the intensity in the anterior chamber was greater for the glaucomatous eye (R) than the control one (L) in the first 10 min
after systemic Gd-DTPA administration.
K.C. Chan et al. / Experimental Eye Research 87 (2008) 334–341
337
Fig. 3. Time course of T1W signal enhancement of the vitreous body (left column) and the anterior chamber (right column) of both eyes before and at 10–80 min following GdDTPA administration. The anterior chamber enhanced substantially over the first 40–60 min after systemic Gd-DTPA administration in both eyes of all animals at Day 5 (Group 1)
(top row) and Week 8 (Group 2) (bottom row). The vitreous body did not enhance in the control eye, yet there was a progressive increase in signal intensity in the vitreous body of
the glaucomatous eye. (ANOVA across timeline in glaucomatous (g) and control (c) eyes with xp < 0.05 and xxp < 0.01). Significant difference in the vitreous body was observed
between the glaucomatous and the control eyes at Week 8 (Group 2) (paired t-test, *p < 0.05), and was marginally observed at Day 5 (Group 1) (paired t-test, p < 0.07 for the later 2
time points). No apparent difference between the two groups was observed in the signal changes in the glaucomatous or control eye.
10.37 2.08 mm3, respectively, between glaucomatous and control
eyes, p < 0.05) and Group 2 (13.36 4.18 mm3 vs 11.75 3.67 mm3,
respectively, between glaucomatous and control eyes, p < 0.05).
Note that there was no significant difference in the eye dimensions
of the same side between two groups.
4. Discussions
In the present study we characterized the ocular transport in
a rat model of chronic glaucoma. The results presented here constitute the first report in assessing the leakage at the aqueous–
vitreous interface in glaucoma by Gd-DTPA enhanced MRI. In the
current glaucoma model, obstruction of aqueous humor outflow is
the primary mechanism of pressure elevation analogous to the
pathogenesis of human POAG. When a persistent elevation of IOP
by 1.6 times was maintained in the rat eye, a 3% retinal ganglion cell
loss per week was documented across the 8-week experimental
period (Li et al., 2006a,b). Our present results suggested an alteration of the typical aqueous outflow in the glaucomatous eye that
might explain the changing biochemical compositions in the anterior chamber and vitreous body (Hu et al., 2002; Lee et al., 1990;
Levkovitch-Verbin et al., 2002a; Ngumah et al., 2006; Prasanna
et al., 2005). These changes might provide some insights in the
cascades of neurodegenerative processes in the retina and optic
nerve.
The aqueous humor proteins have been identified as suspected
participants in the obstruction of the aqueous humor outflow
network from the eye resulting in elevated IOP in POAG (Barsotti
et al., 1992). Protein concentration in the anterior chamber of
normal eyes is determined by three factors: (a) the rate of protein
entry into the aqueous humor, (b) the removal rate by bulk flow of
aqueous humor, and (c) the anterior chamber volume (Murray and
Bartels, 1993). It was known that the blood–aqueous barrier
bordered the posterior edge of the posterior chamber and had
a complex structure that included a posterior epithelial portion
which restricted penetration into the posterior chamber, and an
anterior endothelial portion which restricted leakage into the anterior chamber (Galluzzi et al., 2003). Breakdown of blood–aqueous
barrier resulted in an increase in the aqueous protein level and the
presence of cytokines and other inflammatory mediators in the
aqueous humor (Freddo et al., 2006; Kolodny et al., 2002). For instance, the elevation of aqueous vascular endothelial growth factor
(VEGF) concentration which was known to induce hyperpermeability of microvessels (Alikacem et al., 2000) in various
glaucomas could be attributed primarily to increased production
locally (Aiello et al., 1994). Elevated levels of aqueous humor
endothelin-1 (ET-1) as seen in glaucoma might also have detrimental effects on aqueous humor dynamics such as the contractile
effects on trabecular meshwork vs ciliary muscle (Prasanna et al.,
2005). These might introduce the increased infiltration of Gd-DTPA
into the anterior chamber in some glaucomatous eyes in the current study.
On the other hand, the T1-weighted signal increase in the vitreous body indicated the leakage of Gd-DTPA at the aqueous–vitreous interface upon aqueous outflow obstruction. The aqueous
flow was responsible for creating a positive pressure within the eye,
while the vitreous body was porous and thus transmitted the IOP
from the anterior chamber throughout the interior of the eye
(Ethier et al., 2004). Under normal circumstances, fluid would flow
freely from the vitreous body to the anterior chamber (Araie and
Maurice, 1991), equalizing the pressure across the lens–iris diaphragm (Francis et al., 2002). The stability of the minimal enhancement in the posterior chamber as shown in the control eye
reinforced the role of bulk flow of aqueous forward through the
pupil as an important factor limiting back-diffusion of solutes delivered to the anterior chamber via this anterior pathway, upon the
338
K.C. Chan et al. / Experimental Eye Research 87 (2008) 334–341
Fig. 4. Windowed and zoomed serial T1WIs of the Week 8 (Group 2) glaucomatous (a) and control (b) eyeballs 10–80 min following systemic Gd-DTPA administration. The
corresponding un-windowed image at 80 min post-administration was also shown on the left side to illustrate the regions of interest for zooming. Different extents of leakiness
from the aqueous–vitreous interface (arrows) into the glaucomatous vitreous body were visible as time went by. No apparent leakage was observed in the control eye.
presence of tight junctions between the posterior epithelial cells of
the iris (Bert et al., 2006). When the IOP was lowered, movement of
fluorescein-labeled dextran fractions from the vitreous body into
the anterior chamber was greater and was thought to be attributable to an increase in the anteriorly directed fluid movement across
the aqueous–vitreous interface (Sugiura and Araie, 1989). As the
IOP was increased in the current glaucoma model, it was likely that
this anteriorly directed fluid movement would be reduced. On the
other hand, the aqueous humor proteins originating from the ciliary body capillaries were also prevented from entering the posterior chamber by the tight junctions at the ciliary body epithelium
(Barsotti et al., 1992). In response to prolonged IOP elevation, the
ciliary epithelium has been shown to undergo atrophy in canine
(Smith et al., 1993). Iris stromal atrophy has also been observed in
DBA/2J mice known to develop glaucoma without laser treatment
(Chang et al., 1999; John et al., 1998). When there’s any rise in
blood–aqueous permeability, the reduced limitation of back-diffusion of solutes might attribute to the posterior transport of GdDTPA from the anterior chamber to the vitreous body together with
the reduced anterior bulk flow. This pattern of leakage of proteins
from the blood supply in the ciliary body/iris complex has been
postulated in experimental newborn rats, when the tight junctions
of the blood–ocular barriers were underdeveloped (Berkowitz
et al., 1998). Although it could not be excluded that the leakage of
Gd-DTPA might be partially coming from laser damage as venous
congestion was thought to be a major contributor to IOP elevation
and retinal damage in the current model, the laser treatment was
performed only on the surface of the eyeball in our model. Further,
in other glaucoma models using laser trabecular photocoagulation
and saline injection into limbal veins, and in DBA/2J mice, anterior
synechiae have been noticed (Chang et al., 1999; Grozdanic et al.,
2003; John et al., 1998; Levkovitch-Verbin et al., 2002b; Morrison
et al., 1997; Ueda et al., 1998). It is possible that anterior synechiae
would accompany with aqueous misdirection, in which the aqueous humor is diverted posteriorly and sequestered in or behind the
vitreous body (Azuara-Blanco et al., 1998; Denis, 2002; Francis
et al., 2002).
It was interesting that there was an increase in anterior chamber
volume in the glaucomatous eyes in both groups after glaucoma
induction. Given the fixed axial eye length upon glaucoma induction, the expansion of the anterior chamber implied a posterior
shift of the lens. In one of the glaucomatous eyes in Group 2, a faster
leakage into the vitreous body was observed compared to the other
diseased eyes of the same group, while a prominent lens depression
was also noted (Fig. 5). In human ocular hypertension, lens depression increased Schlemm’s canal volume and partially prevented its collapse at high levels of IOP (Moses et al., 1982; Van
Buskirk, 1982). Yet at the same time, the iris was pushed backwards
and the pectinate ligament was expanded by the posterior displacement of the detached portion of the ciliary body as observed
K.C. Chan et al. / Experimental Eye Research 87 (2008) 334–341
in models using perfusion of the anterior chamber at elevated
pressures (Carreras et al., 1997). A significant increase in the
number of giant vacuoles was also found in the endothelium of
several blood vessels in the rat ciliary body and the iris root
(McMenamin and al-Shakarchi, 1989). These might also contribute
to the compromise of the aqueous–vitreous barrier integrity
allowing Gd-DTPA to pass into the vitreous body. Proton NMR
spectroscopy has once shown an elevated vitreous lactate level in
a rabbit model of ocular hypertension (Ngumah et al., 2006). It was
also known that free amino acids and total protein levels were
increased in the vitreous body in rat chronic ocular hypertension
(Levkovitch-Verbin et al., 2002a). Our results potentially explained
the routes for the leakage of serum proteins found in the vitreous
body of the glaucomatous eye, as well as the release of proteolytic
enzymes which degraded the proteins into free amino acids upon
chronic intraocular hypertension (Levkovitch-Verbin et al., 2002a).
It has been documented that hyperpermeable optic disc capillaries could be observed in glaucoma patients leading to the leakage of fluoresceins into the vitreous body (Tsukahara, 1978).
Nevertheless, given the observation of the earlier T1-weighted
signal increase in the anterior vitreous body than the preretinal
vitreous as in Fig. 4, it was likely that the Gd-DTPA entered the
vitreous body by leakage at the aqueous–vitreous interface in the
glaucomatous eye rather than the compromise of blood–retinal
barrier integrity at the fundus. In addition, it was assumed that
there was no ischemic change in the retina in our model due to the
constant ocular blood flow at mild IOP elevation (Ethier et al., 2004;
Li et al., 2006a,b) and the intact outer nuclear layer or constant total
retinal thickness even at Week 8 after glaucoma induction (Li et al.,
2006b; Yu et al., 2006). Laser-induced damage to the peripheral
retina was also not detected by light microscopy in a similar model
of chronic ocular hypertension (Grozdanic et al., 2003). The validity
of our model in the current study has been verified by measuring
the IOPs of the glaucomatous and control eyes in the Week 8 group
using a calibrated tonometer (Tonopen-XL, Mentor, Norwell, MA,
USA), showing the baseline IOPs of 13.62 0.32 mmHg and
13.83 0.32 mmHg for the glaucomatous and control eyes, respectively,
before
laser
treatment
(p ¼ 0.71),
and
22.74 0.79 mmHg and 13.05 0.50 mmHg, respectively, 10 days
after laser treatment (p < 0.0001), as in consistency with the values
demonstrated by other studies of our group at the similar time
point (Chan et al., 2007, 2008; Fu et al., 2008a,b; Li et al., 2006a,b).
Gd-DTPA enhanced MRI was also performed at Week 18 on three of
the Group 2 animals scanned, and similar pattern of Gd-DTPA
leakage was observed as in Groups 1 and 2 (data not shown). No
339
statistical difference was observed in the signal changes in the
glaucomatous eyes between two groups, which was in agreement
with the fact that a persistent IOP elevation of 1.6 times above
normal was maintained in our model across the 8-week experimental period (Li et al., 2006a,b). A positive correlation was observed between IOP and T1-weighted signal increase in the
vitreous body of the glaucomatous and control eyes at the end-time
point of the experiment (80 min after Gd-DTPA injection) in Group
2 (r2 ¼ 0.54, p < 0.01, N ¼ 6 glaucoma þ 6 control in 6 animals),
assuming a consistent elevation of IOP between Week 2 and Week 8
after laser treatment. These suggested that a higher IOP might give
rise to a larger extent of leakage in the vitreous body. To further
understand the correlation between distinct IOP changes in glaucoma and the extent of leak, future studies would employ Gd-DTPA
enhanced MRI to other glaucoma models that would produce distinctly different extents of ocular hypertension to the current
model (Morrison et al., 1997). We may also apply drug administration (Avila et al., 2003) to alter the IOP in the animals at different
disease stages and evaluate the glaucoma progress in the anterior
visual components.
Gd-DTPA is a water soluble MRI contrast agent with a molecular
weight similar to drugs of interest in ophthalmologic delivery, and
is a standard pharmaceutical agent which is FDA approved for use
in humans. It has been shown that unlike fluorescein, Gd-DTPA is
not actively transported from the chambers, has a well-defined
pharmacokinetic profile, is not metabolized, does not bind to
plasma proteins, will cross blood–ocular barrier only after disruption of tight cell junctions, and can be used to provide an accurate
measure of passive blood–ocular barrier permeability (Trick et al.,
2005). Even if optic media opacity occurs, MRI can provide direct
inner-depth imaging for early screening purposes. Based on the
findings in the current study, it is likely that the Gd-DTPA enhanced
MRI can be translated to study human POAG and provide a diagnostic tool to evaluate the integrity of the aqueous–vitreous
barrier in humans.
5. Conclusion
The present study demonstrated the values of MRI in providing
a means for direct non-invasive visualization of physiological kinetics in the anterior chamber and vitreous body of the in vivo eye.
Gd-DTPA enhanced MRI showed a progressive increase in the vitreous body of the glaucomatous eye but not the control eye, while
an earlier Gd-DTPA transport was found in the anterior chamber of
the glaucomatous eye. In the rat model of chronic glaucoma, an
Fig. 5. Leakage of Gd-DTPA into the vitreous body appeared faster in one of the 6 glaucomatous eyes in Group 2. A prominent lens depression was observed at the same time along
the direction pointed by the arrow.
340
K.C. Chan et al. / Experimental Eye Research 87 (2008) 334–341
array of changes occurred that altered both barrier permeability
and aqueous humor flow and kinetics, leading to the abnormal
enhancements in the anterior chamber and the vitreous body. Our
findings may explain the sources of changing biochemical compositions in the chamber components of the glaucomatous eye,
which may implicate the cascades of neurodegenerative processes
in the retina and the optic nerve. In addition, Gd-DTPA enhanced
MRI provided an in vivo, longitudinal and global means to investigate the abnormalities in the rat model of chronic glaucoma,
and possibly in human POAG. It may also be useful for early
quantitative evaluation of drug treatment effects in patients with
chronic glaucoma, and may complement the conventional techniques in examining the glaucomatous visual components.
Acknowledgments
This work was supported in part by Hong Kong Research Grant
Council and The University of Hong Kong CRCG grant.
References
Aiello, L.P., Avery, R.L., Arrigg, P.G., Keyt, B.A., Jampel, H.D., Shah, S.T., Pasquale, L.R.,
Thieme, H., Iwamoto, M.A., Park, J.E., et al., 1994. Vascular endothelial growth
factor in ocular fluid of patients with diabetic retinopathy and other retinal
disorders. N. Engl. J. Med. 331, 1480–1487.
Alikacem, N., Yoshizawa, T., Nelson, K.D., Wilson, C.A., 2000. Quantitative MR imaging study of intravitreal sustained release of VEGF in rabbits. Investig. Ophthalmol. Vis. Sci. 41, 1561–1569.
Araie, M., Maurice, D.M., 1991. The loss of fluorescein, fluorescein glucuronide and
fluorescein isothiocyanate dextran from the vitreous by the anterior and retinal
pathways. Exp. Eye Res. 52, 27–39.
Avila, M.Y., Mitchell, C.H., Stone, R.A., Civan, M.M., 2003. Noninvasive assessment of
aqueous humor turnover in the mouse eye. Investig. Ophthalmol. Vis. Sci. 44,
722–727.
Azuara-Blanco, A., Katz, L.J., Gandham, S.B., Spaeth, G.L., 1998. Pars plana tube insertion of aqueous shunt with vitrectomy in malignant glaucoma. Arch. Ophthalmol. 116, 808–810.
Barsotti, M.F., Bartels, S.P., Freddo, T.F., Kamm, R.D., 1992. The source of protein in
the aqueous humor of the normal monkey eye. Investig. Ophthalmol. Vis. Sci.
33, 581–595.
Berkowitz, B.A., Roberto, K.A., Penn, J.S., 1998. The vitreous protein concentration is
increased prior to neovascularization in experimental ROP. Curr. Eye Res. 17,
218–221.
Berkowitz, B.A., Roberts, R., Luan, H., Peysakhov, J., Mao, X., Thomas, K.A., 2004.
Dynamic contrast-enhanced MRI measurements of passive permeability
through blood retinal barrier in diabetic rats. Investig. Ophthalmol. Vis. Sci. 45,
2391–2398.
Berkowitz, B.A., Sato, Y., Wilson, C.A., de Juan, E., 1991. Blood-retinal barrier
breakdown investigated by real-time magnetic resonance imaging after gadolinium-diethylenetriaminepentaacetic acid injection. Investig. Ophthalmol. Vis.
Sci. 32, 2854–2860.
Bert, R.J., Caruthers, S.D., Jara, H., Krejza, J., Melhem, E.R., Kolodny, N.H., Patz, S.,
Freddo, T.F., 2006. Demonstration of an anterior diffusional pathway for solutes
in the normal human eye with high spatial resolution contrast-enhanced dynamic MR imaging. Investig. Ophthalmol. Vis. Sci. 47, 5153–5162.
Camelo, S., Shanley, A.C., Voon, A.S., McMenamin, P.G., 2004. An intravital and
confocal microscopic study of the distribution of intracameral antigen in the
aqueous outflow pathways and limbus of the rat eye. Exp. Eye Res. 79, 455–464.
Carreras, F.J., Porcel, D., Gonzalez-Caballero, F., 1997. Expanding forces in aqueous
outflow pathways of a nonaccommodating mammal: an approach via comparative dynamic morphology. Comp. Biochem. Physiol. A Physiol. 117, 197–209.
Chan, H.C., Chang, R.C., Koon-Ching Ip, A., Chiu, K., Yuen, W.H., Zee, S.Y., So, K.F.,
2007. Neuroprotective effects of Lycium barbarum Lynn on protecting retinal
ganglion cells in an ocular hypertension model of glaucoma. Exp. Neurol. 203,
269–273.
Chan, K.C., Fu, Q.L., Hui, E.S., So, K.F., Wu, E.X., 2008. Evaluation of the retina and
optic nerve in a rat model of chronic glaucoma using in vivo manganese-enhanced magnetic resonance imaging. Neuroimage 40, 1166–1174.
Chang, B., Smith, R.S., Hawes, N.L., Anderson, M.G., Zabaleta, A., Savinova, O.,
Roderick, T.H., Heckenlively, J.R., Davisson, M.T., John, S.W., 1999. Interacting loci
cause severe iris atrophy and glaucoma in DBA/2J mice. Nat. Genet. 21, 405–409.
Cheng, H., Nair, G., Walker, T.A., Kim, M.K., Pardue, M.T., Thule, P.M., Olson, D.E.,
Duong, T.Q., 2006. Structural and functional MRI reveals multiple retinal layers.
Proc. Natl. Acad. Sci. U.S.A. 103, 17525–17530.
De Groot, V., Hubert, M., Van Best, J.A., Engelen, S., Van Aelst, S., Tassignon, M.J.,
2003. Lack of fluorophotometric evidence of aqueous–vitreous barrier disruption after posterior capsulorhexis. J. Cataract Refract. Surg. 29, 2330–2338.
Denis, H.M., 2002. Anterior lens capsule disruption and suspected malignant
glaucoma in a dog. Vet. Ophthalmol. 5, 79–83.
Ethier, C.R., Johnson, M., Ruberti, J., 2004. Ocular biomechanics and biotransport.
Annu. Rev. Biomed. Eng. 6, 249–273.
Francis, B.A., Wong, R.M., Minckler, D.S., 2002. Slit-lamp needle revision for aqueous
misdirection after trabeculectomy. J. Glaucoma 11, 183–188.
Freddo, T.F., Patz, S., Arshanskiy, Y., 2006. Pilocarpine’s effects on the blood–aqueous
barrier of the human eye as assessed by high-resolution, contrast magnetic
resonance imaging. Exp. Eye Res. 82, 458–464.
Fu, Q.L., Hu, B., Wu, W., Pepinsky, R.B., Mi, S., So, K.F., 2008a. Blocking LINGO-1
function promotes retinal ganglion cell survival following ocular hypertension
and optic nerve transection. Investig. Ophthalmol. Vis. Sci. 49, 975–985.
Fu, Q.L., Wu, W., Wang, H., Li, X., Lee, V.W., So, K.F., 2008b. Up-regulated endogenous
erythropoietin/erythropoietin receptor system and exogenous erythropoietin
rescue retinal ganglion cells after chronic ocular hypertension. Cell. Mol. Neurobiol. 28, 317–329.
Galluzzi, P., Cerase, A., Hadjistilianou, T., De Francesco, S., Toti, P., Vallone, I.M.,
Filosomi, G., Monti, L., Bracco, S., Gennari, P., Ginanneschi, C., Venturi, C., 2003.
Retinoblastoma: abnormal gadolinium enhancement of anterior segment of
eyes at MR imaging with clinical and histopathologic correlation. Radiology
228, 683–690.
Grozdanic, S.D., Betts, D.M., Sakaguchi, D.S., Allbaugh, R.A., Kwon, Y.H., Kardon, R.H.,
2003. Laser-induced mouse model of chronic ocular hypertension. Investig.
Ophthalmol. Vis. Sci. 44, 4337–4346.
Hu, D.N., Ritch, R., Liebmann, J., Liu, Y., Cheng, B., Hu, M.S., 2002. Vascular endothelial growth factor is increased in aqueous humor of glaucomatous eyes. J.
Glaucoma 11, 406–410.
Jacob, T.J., Civan, M.M., 1996. Role of ion channels in aqueous humor formation. Am.
J. Physiol. 271, C703–C720.
John, S.W., Smith, R.S., Savinova, O.V., Hawes, N.L., Chang, B., Turnbull, D.,
Davisson, M., Roderick, T.H., Heckenlively, J.R., 1998. Essential iris atrophy,
pigment dispersion, and glaucoma in DBA/2J mice. Investig. Ophthalmol. Vis.
Sci. 39, 951–962.
Johnson, M., 2006. What controls aqueous humour outflow resistance? Exp. Eye
Res. 82, 545–557.
Kaufman, P.L., 1999. Nitric-oxide synthase and neurodegeneration/neuroprotection.
Proc. Natl. Acad. Sci. U.S.A. 96, 9455–9456.
Kim, H., Lizak, M.J., Tansey, G., Csaky, K.G., Robinson, M.R., Yuan, P., Wang, N.S.,
Lutz, R.J., 2005. Study of ocular transport of drugs released from an intravitreal implant using magnetic resonance imaging. Ann. Biomed. Eng. 33,
150–164.
Kim, S.H., Csaky, K.G., Wang, N.S., Lutz, R.J., 2008. Drug elimination kinetics following subconjunctival injection using dynamic contrast-enhanced magnetic
resonance imaging. Pharm. Res. 25, 512–520.
Kim, S.H., Galban, C.J., Lutz, R.J., Dedrick, R.L., Csaky, K.G., Lizak, M.J., Wang, N.S.,
Tansey, G., Robinson, M.R., 2007. Assessment of subconjunctival and intrascleral
drug delivery to the posterior segment using dynamic contrast-enhanced
magnetic resonance imaging. Investig. Ophthalmol. Vis. Sci. 48, 808–814.
Kolodny, N.H., Freddo, T.F., Lawrence, B.A., Suarez, C., Bartels, S.P., 1996. Contrastenhanced magnetic resonance imaging confirmation of an anterior protein
pathway in normal rabbit eyes. Investig. Ophthalmol. Vis. Sci. 37, 1602–1607.
Kolodny, N.H., Goode, S.T., Ryan, W., Freddo, T.F., 2002. Evaluation of therapeutic
effectiveness using MR imaging in a rabbit model of anterior uveitis. Exp. Eye
Res. 74, 483–491.
Kuryshova, N.I., Vinetskaia, M.I., Erichev, V.P., Artamonov, V.P., Uspenskaia, A.P.,
1998. Permeability of blood–aqueous humor barrier in primary open-angle
glaucoma. Vestn. Oftalmol. 114, 10–13.
Lee, I.S., Yu, Y.S., Kim, D.M., Youn, D.H., Kim, J.Q., 1990. Detection of specific proteins
in the aqueous humor in primary open-angle glaucoma. Korean J. Ophthalmol.
4, 1–4.
Levkovitch-Verbin, H., Martin, K.R., Quigley, H.A., Baumrind, L.A., Pease, M.E.,
Valenta, D., 2002a. Measurement of amino acid levels in the vitreous humor of
rats after chronic intraocular pressure elevation or optic nerve transection. J.
Glaucoma 11, 396–405.
Levkovitch-Verbin, H., Quigley, H.A., Martin, K.R., Valenta, D., Baumrind, L.A.,
Pease, M.E., 2002b. Translimbal laser photocoagulation to the trabecular
meshwork as a model of glaucoma in rats. Investig. Ophthalmol. Vis. Sci. 43,
402–410.
Leydolt, C., Findl, O., Drexler, W., 2007. Effects of change in intraocular pressure on
axial eye length and lens position. Eye.
Li, R.S., Chen, B.Y., Tay, D.K., Chan, H.H., Pu, M.L., So, K.F., 2006a. Melanopsin-expressing retinal ganglion cells are more injury-resistant in a chronic ocular
hypertension model. Investig. Ophthalmol. Vis. Sci. 47, 2951–2958.
Li, R.S., Tay, D.K., Chan, H.H., So, K.F., 2006b. Changes of retinal functions following
the induction of ocular hypertension in rats using argon laser photocoagulation.
Clin. Experiment. Ophthalmol. 34, 575–583.
Li, S.K., Jeong, E.K., Hastings, M.S., 2004a. Magnetic resonance imaging study of
current and ion delivery into the eye during transscleral and transcorneal
iontophoresis. Investig. Ophthalmol. Vis. Sci. 45, 1224–1231.
Li, S.K., M, J.L., Jeong, E.K., 2008a. MRI in ocular drug delivery. NMR Biomed..
Li, S.K., Molokhia, S.A., Jeong, E.K., 2004b. Assessment of subconjunctival delivery
with model ionic permeants and magnetic resonance imaging. Pharm. Res. 21,
2175–2184.
Li, Y., Cheng, H., Duong, T.Q., 2008b. Blood-flow magnetic resonance imaging of the
retina. Neuroimage 39, 1744–1751.
Manfre, L., Midiri, M., Giuffre, G., Mangiameli, A., Cardella, G., Ponte, F., De Maria, M.,
Lagalla, R., 1997. Blood-ocular barrier damage: use of contrast-enhanced MRI.
Eur. Radiol. 7, 110–114.
K.C. Chan et al. / Experimental Eye Research 87 (2008) 334–341
McMenamin, P.G., al-Shakarchi, M.J., 1989. The effect of various levels of intraocular
pressure on the rat aqueous outflow system. J. Anat. 162, 67–82.
Mermoud, A., Baerveldt, G., Minckler, D.S., Prata Jr., J.A., Rao, N.A., 1996. Aqueous
humor dynamics in rats. Graefes Arch. Clin. Exp. Ophthalmol. 234 (Suppl. 1),
S198–S203.
Molokhia, S.A., Jeong, E.K., Higuchi, W.I., Li, S.K., 2007. Examination of penetration
routes and distribution of ionic permeants during and after transscleral iontophoresis with magnetic resonance imaging. Int. J. Pharm. 335, 46–53.
Molokhia, S.A., Jeong, E.K., Higuchi, W.I., Li, S.K., 2008. Examination of barriers and
barrier alteration in transscleral iontophoresis. J. Pharm. Sci. 97, 831–844.
Morrison, J.C., Fraunfelder, F.W., Milne, S.T., Moore, C.G., 1995. Limbal microvasculature of the rat eye. Investig. Ophthalmol. Vis. Sci. 36, 751–756.
Morrison, J.C., Moore, C.G., Deppmeier, L.M., Gold, B.G., Meshul, C.K., Johnson, E.C.,
1997. A rat model of chronic pressure-induced optic nerve damage. Exp. Eye
Res. 64, 85–96.
Moses, R.A., Etheridge, E.L., Grodzki Jr., W.J., 1982. The effect of lens depression on
the components of outflow resistance. Investig. Ophthalmol. Vis. Sci. 22, 37–44.
Murray, D.L., Bartels, S.P., 1993. The relationship between aqueous humor flow and
anterior chamber protein concentration in rabbits. Investig. Ophthalmol. Vis.
Sci. 34, 370–376.
Ngumah, Q.C., Buchthal, S.D., Dacheux, R.F., 2006. Longitudinal non-invasive proton
NMR spectroscopy measurement of vitreous lactate in a rabbit model of ocular
hypertension. Exp. Eye Res. 83, 390–400.
Ozaki, L., 1984. The barrier function of the posterior capsule. J. Am. Intraocul. Implant. Soc. 10, 182–184.
Patz, S., Bert, R.J., Frederick, E., Freddo, T.F., 2007. T(1) and T(2) measurements of the
fine structures of the in vivo and enucleated human eye. J. Magn. Reson. Imaging 26, 510–518.
Prasanna, G., Hulet, C., Desai, D., Krishnamoorthy, R.R., Narayan, S., Brun, A.M.,
Suburo, A.M., Yorio, T., 2005. Effect of elevated intraocular pressure on endothelin-1 in a rat model of glaucoma. Pharmacol. Res. 51, 41–50.
Quigley, H.A., Broman, A.T., 2006. The number of people with glaucoma worldwide
in 2010 and 2020. Br. J. Ophthalmol. 90, 262–267.
341
Raviola, G., Raviola, E., 1978. Intercellular junctions in the ciliary epithelium. Investig. Ophthalmol. Vis. Sci. 17, 958–981.
Sato, Y., Berkowitz, B.A., Wilson, C.A., de Juan Jr., E., 1992. Blood-retinal barrier
breakdown caused by diode vs argon laser endophotocoagulation. Arch. Ophthalmol. 110, 277–281.
Shen, Q., Cheng, H., Pardue, M.T., Chang, T.F., Nair, G., Vo, V.T., Shonat, R.D., Duong, T.
Q., 2006. Magnetic resonance imaging of tissue and vascular layers in the cat
retina. J. Magn. Reson. Imaging 23, 465–472.
Smith, R., Peiffer, R., Wilcock, B., 1993. Some aspects of the pathology of canine
glaucoma. Prog. Vet. Comp. Ophthalmol. 3, 16–28.
Sugiura, Y., Araie, M., 1989. Effects of intraocular pressure change on movement of
FITC-dextran across vitreous–aqueous interface. Jpn. J. Ophthalmol. 33, 441–450.
Trick, G.L., Liggett, J., Levy, J., Adamsons, I., Edwards, P., Desai, U., Tofts, P.S.,
Berkowitz, B.A., 2005. Dynamic contrast enhanced MRI in patients with diabetic
macular edema: initial results. Exp. Eye Res. 81, 97–102.
Tsukahara, S., 1978. Hyperpermeable disc capillaries in glaucoma. Adv. Ophthalmol.
35, 65–72.
Ueda, J., Sawaguchi, S., Hanyu, T., Yaoeda, K., Fukuchi, T., Abe, H., Ozawa, H., 1998.
Experimental glaucoma model in the rat induced by laser trabecular photocoagulation after an intracameral injection of India ink. Jpn. J. Ophthalmol. 42,
337–344.
Van Buskirk, E.M., 1980. The canine eye: lens depression and aqueous outflow.
Investig. Ophthalmol. Vis. Sci. 19, 789–792.
Van Buskirk, E.M., 1982. Anatomic correlates of changing aqueous outflow facility in
excised human eyes. Investig. Ophthalmol. Vis. Sci. 22, 625–632.
Wilson, C.A., Berkowitz, B.A., Funatsu, H., Metrikin, D.C., Harrison, D.W., Lam, M.K.,
Sonkin, P.L., 1995. Blood-retinal barrier breakdown following experimental
retinal ischemia and reperfusion. Exp. Eye Res. 61, 547–557.
WoldeMussie, E., Ruiz, G., Wijono, M., Wheeler, L.A., 2001. Neuroprotection of
retinal ganglion cells by brimonidine in rats with laser-induced chronic ocular
hypertension. Invest. Ophthalmol. Vis. Sci. 42, 2849–2855.
Yu, S., Tanabe, T., Yoshimura, N., 2006. A rat model of glaucoma induced by episcleral vein ligation. Exp. Eye Res. 83, 758–770.