Elsevier

NeuroImage

Volume 32, Issue 3, September 2006, Pages 1195-1204
NeuroImage

Differential sensitivity of in vivo and ex vivo diffusion tensor imaging to evolving optic nerve injury in mice with retinal ischemia

https://doi.org/10.1016/j.neuroimage.2006.04.212Get rights and content

Abstract

Decreased axial (λ||) and increased radial (λ) diffusivity have been shown to reflect axonal and myelin injury respectively. In the present study, evolving white matter injury within the optic nerves of mice with retinal ischemia was examined by in vivo and ex vivo measurements of λ|| and λ. The results show that at 3 days after retinal ischemia, a 33% decrease in vivo and a 38% decrease ex vivo in λ|| without change in λ was observed in the injured optic nerve compared to the control, suggestive of axonal damage without myelin injury. At 14 days, both in vivo and ex vivo measured λ increased significantly to 220–240% of the control level in the injured optic nerve suggestive of myelin damage. In contrast, the axonal injury that was clearly detected in vivo as a significantly decreased λ|| (33% decrease) was not as clearly detected by ex vivo λ|| (17% decrease). The current findings suggest that ex vivo λ is comparable to in vivo λ in detecting myelin injury. However, the structural changes resulting from axonal damage causing the decreased in vivo λ|| may not be preserved ex vivo in the fixed tissues. Despite the accurate depiction of the pathology using λ|| and λ in vivo, the use of ex vivo λ|| to extrapolate the status of axonal injury in vivo would require further investigation.

Introduction

Tissue fixation is a ubiquitous laboratory practice, and it has become increasingly common to obtain magnetic resonance images (MRI) from fixed tissues (Ahrens et al., 1998, Bonny and Renou, 2002, Holmes et al., 2000, Hsu et al., 1998, Jacobs et al., 1999, Mori et al., 1999, Mori et al., 2001, Pfefferbaum et al., 2004, Wedeen et al., 2001). Recent applications of diffusion tensor imaging (DTI) to fixed tissue demonstrated that it is possible to obtain data with high signal-to-noise ratio (SNR) at high spatial resolution without motion artifact as compared with in vivo studies (Mori et al., 1999, Mori et al., 2001). Postmortem brain specimens from patients with Alzheimer's disease (Bronge et al., 2002) or multiple sclerosis (Schmierer et al., 2003, Schmierer et al., 2004) have been examined using MRI to identify neuropathology.

Although increased T2 and decreased magnetization transfer ratio have shown high correlation to histological abnormalities in white matter, these MRI measurements do not differentiate the underlying axonal and myelin damage (Meier et al., 2004). To improve the specificity of MRI to underlying pathology, the directional diffusivities derived from DTI were separated to components describing water movement along (λ||, axial diffusivity) and across (λ, radial diffusivity) the white matter tract. It has been demonstrated in living central and peripheral nervous systems that axonal injury in white matter results in reduced λ|| while myelin damage increases λ (Arfanakis et al., 2002, Beaulieu et al., 1996, Kim et al., 2006, Song et al., 2002, Song et al., 2003, Sun et al., 2006, Thomalla et al., 2004). However, application of directional diffusivity to fixed specimens has only been reported recently in studies examining the effect of demyelination on λ (Song et al., 2005).

Many factors may alter the water diffusion characteristics in formalin-fixed specimens. For example, decrease in tissue temperature (Sun et al., 2003, Sun et al., 2005), cessation of cellular transport (Le Bihan, 1995, van der Toorn et al., 1996) and possible change of cellular membrane permeability (Benga et al., 1986, Gold and Widnell, 1976, Ormerod et al., 1993, Sehy et al., 2002) in fixed tissues can lead to changes in the measured DTI parameters. Thus, careful comparison of in vivo and ex vivo diffusion properties is crucial in establishing the validity and relevance of MRI of fixed specimens. Some preliminary findings suggest that intravoxel anisotropy and inter-voxel coherence of water diffusion are maintained in tissue after fixation (Crespigny et al., 2005, Guilfoyle et al., 2003, Sun et al., 2003, Sun et al., 2005). A 50–70% decrease in mean apparent diffusion coefficient (ADC) after fixation was also reported (Sun et al., 2003, Sun et al., 2005). The decrease in ADC is not uniform across the entire brain. Different regional changes in diffusion magnitude after fixation result in an altered image contrast of ex vivo ADC maps. This alteration has resulted in the loss of sensitivity using ADC to detect an area of acute stroke after fixation (Sun et al., 2005).

To test the utility of λ|| and λ as well as to evaluate the general diffusion characteristics in injured white matter of fixed specimens, the mouse model of transient retinal ischemia (Kawai et al., 2001, Rosenbaum et al., 2001, Song et al., 2003) was employed in this study. The distinct pattern of optic nerve (ON) injury resulting from retinal ischemia, i.e., acute axonal damage followed by coexistence of axonal and myelin damage (Adachi et al., 1996, Song et al., 2003), makes it an ideal model to evaluate both types of injury. Serial examinations of mice undergoing transient retinal ischemia have confirmed the previously reported time course of the injury progression. Two time points were selected representing two different pathological states based on the in vivo DTI findings. Histological analyses after ex vivo DTI were performed to verify the DTI findings.

Section snippets

Retinal ischemia

Male Swiss Webster mice, 6–8 weeks of age underwent transient retinal ischemia (Song et al., 2003). Briefly, 100–120 mm Hg intraocular pressure was applied to the right eye of each mouse by inserting into the anterior chamber a 32-gauge needle connecting to a saline reservoir placed above the eye for 1 h. Reperfusion started immediately after removal of the cannula. The left eye, which was not cannulated, served as the control.

Eight mice were used to evaluate the temporal evolution of ON

In vivo longitudinal measurements of λ|| and λ

Optic nerve degeneration following transient retinal ischemia was evaluated longitudinally over a 21-day period (Fig. 2). As previously published (Song et al., 2003), a significant decrease of λ|| suggestive of axonal injury was seen at 3 days after retinal ischemia. The extent of the reduced λ|| was maintained throughout the entire time course. In contrast, λ was not affected on day 3 but a statistically significant elevation of λ was seen at day 7 and reached a plateau by day 14 (Fig. 2),

Discussion

In this study, the evolution of ON injury was examined both in vivo and ex vivo using DTI. The feasibility of using λ|| and λ as surrogate markers to detect axonal and myelin damage in paraformaldehyde fixed ON was extensively examined in two different settings of injury: (a) axonal injury without myelin damage (decreased λ|| with normal λ at 3 days) and (b) the coexistence of both axonal and myelin damage (decreased λ|| and increased λ at 14 days). At 3 days after ischemia, a comparable

Acknowledgments

This study was supported in part by the National Multiple Sclerosis Society (RG 3376, and CA 1012), NIH R01-NS047592, USUHS grant G170SX, and the Washington University Small Animal Imaging Resource (WUSAIR) (NIH: R24-CA83060). AHC was supported by the Manny and Rosalyn Rosenthal–Dr. John L. Trotter MS Center Chair in Neuroimmunology.

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