Elsevier

NeuroImage

Volume 44, Issue 3, 1 February 2009, Pages 611-619
NeuroImage

Fixation, not death, reduces sensitivity of DTI in detecting optic nerve damage

https://doi.org/10.1016/j.neuroimage.2008.10.032Get rights and content

Abstract

The changes of directional diffusivities derived from diffusion tensor imaging (DTI), i.e. decreased axial diffusivity (λ||) and increased radial diffusivity (λ), have shown significant correlation with axonal and myelin damage, respectively. However, after formalin fixation, reduced sensitivity of λ|| in detecting axonal damage in tissue has raised the concern of applying DTI ex vivo. In order to distinguish whether death or the fixation process diminishes the sensitivity of DTI in detecting lesions, in vivo, pre-fixed postmortem, and fixed postmortem DTI were conducted on mouse optic nerves 3 and 14 days after transient retinal ischemia. Our data showed that, from in vivo to pre-fixed postmortem, λ|| and λ decreased by 50 to 70% in both healthy and injured optic nerves (3 and 14 day injury). From pre-fixed postmortem to fixed postmortem, λ|| and λ decreased by 40 to 50% in normal and 3-day injured optic nerves, but only by 15 to 25% in 14-day injured optic nerves. Consequently, for the 14-day injured optic nerves, the differences between healthy and injured nerves were not preserved after fixation: the 40% decreased λ|| and 200% increased λ in injured nerves as compared to the normal nerves were measured in vivo and pre-fixed postmortem, but after the fixation process, 300% increased λ and insignificant changes in λ|| were found in injured nerves as compared to the normal nerves. This study clarified that fixation process, but not death, could change the sensitivity of DTI in detecting injury.

Introduction

Water diffusion properties quantified by diffusion tensor imaging (DTI) are sensitive in detecting various types and degrees of the damage to the central nervous system. For example, the mean diffusivity decreased within minutes after cerebral ischemia, which was more sensitive to insults than most other MR imaging modalities (Muir et al., 2006, Redgrave et al., 2007). The reduction in axial diffusivity (λ||, quantifying the water diffusivity along the nerve fibers) and the increase in radial diffusivity (λ, quantifying the water diffusivity across the nerve fibers) have shown significant correlation with axonal and myelin damage, respectively (Budde et al., 2007, Kim et al., 2006, Song et al., 2003, Song et al., 2002, Sun et al., 2007, Sun et al., 2006b). In addition, the major eigenvector and diffusion anisotropy provide intrinsic structural information of the nervous system, allowing researchers to conduct non-invasive nerve fiber tracking in three-dimensional space (Mori et al., 2001, Mori and van Zijl, 2002).

Many researchers conducted DTI not only on live subjects, but also on fixed postmortem specimens. The prolonged scanning of imaging postmortem specimens allows researchers to acquire data with increased signal-to-noise ratios and spatial resolutions for characterizing fine structural changes of the subjects (D'Arceuil et al., 2007, Huang et al., 2004, Kroenke et al., 2006). The high quality and resolution of images acquired from ex vivo DTI provide detailed three-dimensional anatomical information for identification of targets of interest to undergo immunohistochemical examinations.

However, little was known regarding the effects of the tissue fixation process in DTI. According to recent studies (Crespigny et al., 2005, Guilfoyle et al., 2003, Sun et al., 2003), the consistent measurements of diffusion anisotropy between in vivo and ex vivo DTI on healthy subjects may attribute to the well preserved tissue structure resulting from the fixation process. The diffusion anisotropy may also be preserved in tissue with delayed fixation, where the fixation process was conducted within 10 h of animal death (Kim et al., 2007). However, as for the injured subjects, the sensitivities of DTI in detecting neurological pathologies could be significantly reduced in the ex vivo study (Sun et al., 2006a, Sun et al., 2005). Specifically, in mouse optic nerves 14 days after transient retinal ischemia, both in vivo and ex vivo DTI showed increased λ in detecting myelin damage, which correlated with immunohistochemistry (Sun et al., 2006a). However, only in vivo, but not ex vivo, λ|| changed significantly enough to provide evidence for axonal damage (Sun et al., 2006a). While ex vivo DTI could provide prolonged scanning for better characterization of changes to the fine structure of the examined samples (D'Arceuil et al., 2007, Huang et al., 2004, Kroenke et al., 2006), the inconsistency between in vivo and ex vivo measurements remains problematic in correctly determining disease by ex vivo DTI.

Despite possible explanations as to why ex vivo λ|| loses sensitivity in detecting axonal damage (Sun et al., 2006a, Sun et al., 2006b, Sun et al., 2005), previous studies did not provide enough evidence to distinguish whether animal death or the fixation process was the primary cause for losing the contrast of λ|| between normal and injured optic nerves. Evaluating the diffusion change from the same animal pre-fixed and fixed postmortem might shed light on how animal death and the fixation process would influence DTI measurements. In this study, Wallerian degeneration to the right optic nerve of each mouse was generated by transient retinal ischemia on the right eye. The left optic nerve remained intact to serve as a control. Three and 14 days after ischemia, DTI was conducted to examine the left and right optic nerves of mice in vivo, pre-fixed postmortem and fixed postmortem DTI. The results could improve our understanding regarding the impact of animal death and the fixation process on DTI in detecting axonal and myelin damages.

Section snippets

Data acquisition

Twelve male Swiss Webster mice, 6–8 weeks of age, underwent one hour transient retinal ischemia preparation (Song et al., 2003, Sun et al., 2006a). Briefly, 100–120 mm Hg intraocular pressure was applied to the right eye of each mouse by inserting a 32-gauge needle connected to a saline reservoir. After 1 h, the reperfusion started immediately after removal of the cannula. The left eye, which was not cannulated, served as the control. On the 3rd day and 14th day after ischemia, respectively (N = 

Results

The left and right optic nerves could be easily identified as two bright dots in RA maps registered with black dots in λ maps (Fig. 1). After ischemia, different pathologies evolved in the injured optic nerve as evidenced by the changes in λ|| and λ in vivo. Specifically, on the 3rd day after ischemia, decrease was seen with λ||, while no change of λ was seen in injured optic nerve suggestive of axonal damage (Fig. 2). In contrast, on the 14th day after ischemia, decrease in λ|| with

Discussions

Derived from in vivo DTI, decreased λ|| and increased λ have demonstrated to be in vivo quantities sensitive in detecting axonal and myelin damage (Song et al., 2003, Song et al., 2002, Song et al., 2005, Sun et al., 2006a, Sun et al., 2006b). However, the discrepancy between in vivo and ex vivo measurements has raised the concern in applying DTI to detect the white matter pathologies in fixed tissues (Sun et al., 2006a, Sun et al., 2005). In this study, the transition from in vivo to ex vivo

Acknowledgments

This study was supported partly by grants provided by NMSS RG-3864 and CA-1012, and NIH R01-NS-054194. We would also like to thank Dr. Sheng-Kwei (Victor) Song for his insightful discussions.

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