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Experimental Eye Research 87 (2008) 334–341 Contents lists available at ScienceDirect Experimental Eye Research journal homepage: www.elsevier.com/locate/yexer 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 336 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. 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