Elsevier

NeuroImage

Volume 40, Issue 3, 15 April 2008, Pages 1166-1174
NeuroImage

Evaluation of the retina and optic nerve in a rat model of chronic glaucoma using in vivo manganese-enhanced magnetic resonance imaging

https://doi.org/10.1016/j.neuroimage.2008.01.002Get rights and content

Abstract

Glaucoma is a neurodegenerative disease of the visual system. While elevated intraocular pressure is considered to be a major risk factor, the primary cause and pathogenesis of the disease are still unclear. This study aims to employ in vivo manganese-enhanced magnetic resonance imaging (MEMRI) to evaluate dynamically the Mn2+ enhancements in the visual components following an induction of ocular hypertension in a rat model of chronic glaucoma. The episcleral and limbal veins were photocoagulated unilaterally in the right eye using an argon laser to maintain a consistent elevation of intraocular pressure by about 1.6 times above the normal level. Two and six weeks after glaucoma induction, MnCl2 solution (50 mM, 3 μL) was injected intravitreally into both eyes, and MEMRI was performed 2 to 5 h after injection. Results showed a delayed increase in T1-weighted signal intensity in the glaucomatous optic nerve at 6 weeks but not 2 weeks after glaucoma induction. In addition, there was an accumulation of Mn2+ ions in the vitreous humour of the glaucomatous eye, with a high concentration of Mn2+ ions at the optic nerve head and the retina. These MEMRI findings may help understand the disease mechanisms, monitor the effect of drug interventions in glaucoma models and complement the conventional techniques in examining the glaucomatous visual components.

Introduction

Glaucoma is a neurodegenerative disease of the visual system characterized by retinal ganglion cell (RGC) death, optic nerve head (ONH) damage and progressive visual field loss (Thanos and Naskar, 2004). It is the second major cause of blindness in the world (Quigley and Broman, 2006). While elevated intraocular pressure (IOP) is considered to be a major risk factor, the primary cause to the disease mechanisms is still unclear (Kaufman, 1999, Morrison et al., 2005). Quantitative assessments of optic nerve (ON) axonal loss or brain changes in experimental models of glaucoma are typically performed postmortem in histological tissue sections (Lindsey et al., 2007, Morrison, 2005) or by electrophysiological techniques (Li et al., 2006a). However, these methods either fail to provide a global view of the brain or do not allow repeated measures for longitudinal studies (van der Linden et al., 2004). To understand the exact mechanisms of glaucomatous changes, there is a need to develop a novel, in vivo and three-dimensional method to investigate into the integrity of the primary visual system longitudinally.

Magnetic resonance imaging (MRI) provides a non-invasive tool to study the structural, metabolic and functional details of the inner depth of the body in vivo. Among the MRI techniques, diffusion tensor imaging has successfully detected and differentiated structurally the axon and myelin degeneration upon an evolving white matter injury in the mouse ON after retinal ischemia (Song et al., 2003, Sun et al., 2006). It has also been shown that the lamina-specific structures and functional responses in the retina can be resolved using Gd-DTPA contrast-enhanced MRI and high-resolution functional MRI (Cheng et al., 2006). Recently, manganese-enhanced magnetic resonance imaging (MEMRI) has been increasingly used to study both structural and functional changes in the central nervous system without reliance on hemodynamic response. It has been used to enhance the contrast in studies of neuroarchitecture (Aoki et al., 2004a), to trace neuronal pathways (Pautler, 2004, Pautler et al., 1998), to detect activated regions in the brain (Silva et al., 2004) and to investigate cerebral ischemic injury (Aoki et al., 2004b, Yang and Wu, 2008). Mn2+ ions are paramagnetic in nature and can shorten the T1 relaxation time of the surrounding water protons. They act as a calcium analogue and enter the intracellular space via L-type voltage-gated calcium channels upon neuronal activation (Pautler, 2006). A fraction of the ions is then sequestered in the endoplasmic reticulum or Golgi apparatus and actively transported along the microtubules via fast axonal transport (Pautler, 2004, Van der Linden et al., 2007, Watanabe et al., 2004). On the other hand, a recent study has demonstrated the independence of electrical activity to Mn2+ uptake in the eye (Bearer et al., 2007), suggestive of alternative channels, e.g. divalent metal transporters (Takeda, 2003) for Mn2+ transport. Glial uptake and diffusion may also contribute to the cerebral pattern in Mn2+ enhancement (Watanabe et al., 2004). In the current study, we aimed to examine the Mn2+ transport in the normal and glaucomatous eyes upon intravitreal injection and to correlate the in vivo results with previous histological findings. These may help optimize the investigation into the integrity of the visual system and the possibility of ocular drug delivery into the glaucomatous eyes.

MEMRI has been used to examine the axonal transport of the central nervous system in rodents. As Mn2+ can access the nervous system intraaxonally without the reliance on hemodynamic response, studies evaluated the axonal transport rate in Alzheimer's disease (Smith et al., 2007) and diabetes (Serrano et al., 2007) and upon drug treatment (Smith et al., 2007). On the other hand, axon degeneration has been demonstrated by the blockade of Mn2+ transport at sites of radiation-induced injury (Ryu et al., 2002) and ON crush (Thuen et al., 2005) upon intravitreal injection. By applying MEMRI, it is also possible to compare the cross-sectional areas in the prechiasmatic regions of the ON induced with optic glioma (Banerjee et al., 2007). In the experimental rat model of chronic glaucoma in our laboratory, chronic IOP elevation induces a 3% RGC loss per week across the 8-week experimental period (Li et al., 2006a). Other reports indicated an early damage from an elevated IOP in the superior regions of the rat ON (Morrison, 2005, Quigley et al., 1987, Wang et al., 2004), while atrophy of large fibers was observed in areas of the ON with mild damage (Johnson et al., 2000, Quigley et al., 1987). It was also suggested that the axoplasmic flow happened to be disturbed upon chronic IOP elevation (Dandona et al., 1991, Johnson et al., 2000, Khosla et al., 1982). In experimental mouse ocular hypertension, the ON mean axon density and total number of axons in the laser-treated eyes were found to be significantly less than in the control eyes (Mabuchi et al., 2003, Mabuchi et al., 2004). Therefore, we hypothesize that there would be a reduction in Mn2+ transport along the glaucomatous ON, especially in the superior regions.

Further, previous studies using high-resolution MEMRI detected layer-specific retinal functional adaptation (Berkowitz et al., 2006), as well as changes in intraretinal signal intensities in rat models of ON injury and choroidal melanoma (Berkowitz et al., 2007, Braun et al., 2007). In addition to the RGC loss, it was shown that ocular hypertension appeared to accompany with ischemia, axonal swelling and mechanical alteration of the laminar layers at the ONH (Gross et al., 2003, Kaufman, 1999). To account for these, we would attempt to test if there would be signal changes in the retina and the ONH between the glaucomatous and the control eyes.

Lastly, Mn2+ has been applied trans-sclerally, transcorneally and intravitreally to evaluate its distribution in the eyeballs for ocular drug delivery (Li et al., 2004, Molokhia et al., 2007). The glaucoma model in the current study involves laser photocoagulation of the episcleral and limbal veins, which contributes to the obstruction of venous outflow in rats (Li et al., 2006a, Morrison et al., 1995). Blockade of this drainage would induce a 1.6-fold increase in IOP (Chan et al., 2007, Fu et al., 2007, Li et al., 2006a, Li et al., 2006b). By employing dynamic MEMRI, we hypothesize that the usual pattern of Mn2+ clearance in the glaucomatous eyeball will be perturbed upon intravitreal injection.

To our knowledge, this is the first attempt to apply dynamic MEMRI to investigate the chronic glaucoma within the rat primary visual system in vivo. This is particularly valuable as we may be able to apply MEMRI to understand the disease mechanisms and resolve the functional loss and recovery of neuronal connectivity (van der Zijden et al., 2007) upon drug treatment (Chan et al., 2007, Fu et al., 2007).

Section snippets

Animal preparation

Sprague–Dawley female rats (250–280 g, 3 months old, N = 15) were reared in a temperature-controlled room subjected to a 12-h light/12-h dark cycle with standard chow and water supply ad libitum. They were divided into 3 groups (see Fig. 1). Except for the control group, the rats were induced for ocular hypertension unilaterally in the right eye by photocoagulation of the episcleral and limbal veins using an argon laser. A second laser treatment in the same settings was applied 7 days later to

Mn2+ transport in prechiasmatic optic nerve

Fig. 2 illustrates the placement of the VOI in one of the prechiasmatic ONs. As shown in Fig. 3, MEMRI signal increased significantly in both sides of the prechiasmatic ONs of all animals over time after Mn2+ injection (ANOVA: p < 0.05). A reduction in the Mn2+ transport was observed at the prechiasmatic region of the glaucomatous ON compared to the contralateral side in the Week 6 model in Group 1, while there was no apparent reduction in the Mn2+ transport in the Week 2 model in Group 2.

Mn2+ transport along the optic nerve

Mn2+ ions have been verified to be uptaken by the RGCs, packed into organelles in the endoplasmic reticulum and transported along microtubules in the ONs (Pautler, 2006, Van der Linden et al., 2007). The rate of signal accumulation at a target location would be expected to depend on the uptake rate of Mn2+ at the injection site, the density of projections and the transport and/or diffusion away from the target location (Leergaard et al., 2003). In the current study, Mn2+ enhancement in ONs was

Acknowledgments

The authors would like to thank Mr. Darrell Li and Mr. Matthew Cheung at Laboratory of Biomedical Imaging and Signal Processing, and Dr. Rachel Li and Ms Phillis Kau at the Department of Anatomy at The University of Hong Kong for technical assistance. This work was supported in part by Hong Kong Research Grant Council and The University of Hong Kong CRCG grant.

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