Elsevier

NeuroImage

Volume 59, Issue 1, 2 January 2012, Pages 363-376
NeuroImage

Magnetic resonance imaging of the mouse visual pathway for in vivo studies of degeneration and regeneration in the CNS

https://doi.org/10.1016/j.neuroimage.2011.07.069Get rights and content

Abstract

Traditionally, depiction of isolated CNS fiber tracts is achieved by histological post mortem studies. As a tracer-dependent strategy, the calcium analog manganese has proved valuable for in vivo imaging of CNS trajectories, particularly in rats. However, adequate protocols in mice are still rare. To take advantage of the numerous genetic mouse mutants that are available to study axonal de- and regeneration processes, a MnCl2-based protocol for high-resolution contrast-enhanced MRI (MEMRI) of the visual pathway in mice acquired on a widely used clinical 3 Tesla scanner was established. Intravitreal application of MnCl2 significantly enhanced T1-weighted contrast and signal intensity along the retino-petal projection enabling its reconstruction in a 3D mode from a maximum intensity projection (MIP) calculated dataset. In response to crush injury of the optic nerve, axonal transport of MnCl2 was diminished and completely blocked proximal and distal to the lesion site, respectively. Conditions of Wallerian degeneration after acute optic nerve injury accelerated Mn2+-enhanced signal fading in axotomized projection areas between 12 and 24 h post-injury. In long-term regeneration studies 12 months after optic nerve injury, the MRI protocol proved highly sensitive and discriminated animals with rare spontaneous axonal regrowth from non-regenerating specimens. Also, structural MRI aspects shared high correlation with histological results in identical animals. Moreover, in a model of chronic neurodegeneration in p50/NF-κB-deficient mice, MnCl2-based neuron-axonal tracing supported by heat map imaging indicated neuropathy of the visual pathway due to atrophy of optic nerve fiber projections. Toxic effects of MnCl2 at MRI contrast-relevant dosages in repetitive administration protocols were ruled out by histological and optometric examinations. At higher dosages, photoreceptors, not retinal ganglion cells, turned out as most susceptible to the well-known toxicity of MnCl2. Our data accentuate in vivo MEMRI of the murine visual system as a highly specific and sensitive strategy to uncover axonal degeneration and restoration processes, even in a functional latent state. We expect MEMRI to be promising for future applications in longitudinal studies on development, aging, or regeneration of CNS projections in mouse models mimicking human CNS pathologies.

Highlights

► Mouse-specific technically innovative, high-sensitive MEMRI protocol. ► Detailed species-specific analysis on tracer concentrations and pharmacokinetics. ► Functional and histological toxicity of MnCl2 in single and serial applications. ► Proof of protocol in CNS models of neurode- and regeneration. ► Implications for long-term studies and use on genetically engineered mice.

Introduction

Axonal pathologies of CNS fiber projections, e.g. after acute spinal cord injury, stroke, or in neurodegenerative diseases, indicate special diagnostic means to address structural alterations at incipiency, and to monitor treatment efficacy or to predict disease progression. Apart from diffusion tensor (DT)-MRI and MR-spectroscopy, which are commonly reserved to peculiar indications, non-invasive MRI sequences are standard in clinical brain imaging, for analysis of local CNS circuits, and volume calculations. However, the concomitantly applied contrasting agent Gadolinium (Gd)-DTPA accumulates only at site of disrupted blood brain barrier (BBB), and often neglects non-inflammatory pathology of single fiber projections. In experimental in vivo paradigms, e.g. in animal models, tracer substances such as biotinylated dextran, Fluorogold, DiI, or horseradish peroxidase can be directly applied to the neuronal soma or to synaptic terminals to label centrifugal or centripetal fiber projections. Neurotropic reporter gene coupled Herpes simplex virus constructs trace trans-synaptic projections when locally administered to the brain region of interest. Such chemical tracing processes always require the sacrifice of animals for subsequent histological examinations as well as time intense software reconstructions of serial sections. An elegant technique for in vivo fiber tract visualization in several species including mice, rats and non-human primates represents the use of Mn2+-enhanced MRI (MEMRI) as pioneered by Watanabe et al., 2001, Watanabe et al., 2004a, Watanabe et al., 2004b, Pautler, 2004, Pautler and Koretsky, 2002, Pautler et al., 1998, Pautler et al., 2003. Meanwhile, their original work has been extended and comparative DTI studies and MEMRI features on acutely injured and chronically degenerating axonal CNS projections are available (Kim et al., 2011, Thuen et al., 2008, Thuen et al., 2009). Neuroanatomically, sensory CNS tracts such as the olfactory, visual and auditory projection have become focus of MEMRI (Pautler and Koretsky, 2002, Watanabe et al., 2001, Watanabe et al., 2008). However, it also unmasked structural alterations in the prefrontal to mesocortical reward-modulating circuitry in dopamine transporter knockout mice that had remained underestimated even by voxel-wise statistical MRI analysis (Zhang et al., 2010). Further genetically modified mouse mutants served to characterize physiological suppositions and pathological impairments of axonal Mn2+ transport. Likewise, altered transport velocities were exemplified in triple transgenic mice mimicking Alzheimer tauopathy (Kim et al., 2011). Bearer et al. (2007) investigated the role of neuronal activity on trans-synaptic Mn2+ propagation and its dependence on kinesin-mediated axonal transport. The activity dependence of Mn2+ propagation was used for fMRI-like MEMRI studies on in vivo mapping of the auditory pathway in which the tonotopic representation of the inferior colliculi under frequency modulated acoustic stimulation was presented in a highly sophisticated manner (Yu et al., 2005). Similar studies could recapitulate the retinotopic structural organization of the superior colliculi (Chan et al., 2011). Most importantly, the chelated FDA-approved Mn2+ derivative manganese dipyridoxyl diphosphate (MnDPDP, Teslascan) is already under clinical application, e.g. for increasing diagnostic sensitivity of MRI on liver and pancreas, and a first experimental study on intravenous application of Teslascan at clinically relevant doses in SD rats and for imaging of the visual pathway are already available (Olsen et al., 2008, Tofts et al., 2010).

Biophysically, paramagnetic Mn2+ and Gd-DTPA augment magnetic resonance contrast mainly by shortening T1 spin-lattice relaxation time (Howles et al., 2010, Mendonca-Dias et al., 1983). As a calcium (Ca2+) analog, Mn2+ is incorporated in neurons by voltage-gated calcium channels and becomes actively transported along intact microtubules of the axonal cytoskeleton, whereas passive diffusion into the tissue is negligible. Its propagation is interrupted both mechanically and toxically, e.g. in injured axonal trajectories (Thuen et al., 2005) and upon cholchicine treatment (Tillet et al., 1993). It accumulates as a function of membrane integrity and cell activation, and is retained intracellulary in vesicles for longer time intervals. MEMRI of distinctive CNS structures has been shown to be feasible in rodents following its systemic intravenous (Boretius et al., 2008), intraperitoneal, or subcutaneous (Watanabe et al., 2002, Watanabe et al., 2004a, Yu et al., 2005) and local, e.g. stereotaxic (Pautler et al., 1998, Watanabe et al., 2004b, Zhang et al., 2010) or aerosolized (Pautler and Koretsky, 2002) administration.

Here, we introduce a novel MEMRI protocol to dissect axonal disorders in mice using T1-weighted sequences. In contrast to recent studies on mice performed with 7.0 up to 11.7 T high resolution magnetic resonance machines (Lindsey et al., 2007, Zhang et al., 2010), images were acquired on a conventional clinical 3 T scanner, thus providing technical accessibility to a much broader audience in neuroscience research. MnCl2 solutions were applied intravitreally (ivit), and time- and concentration-dependent alterations of T1-weighted contrast and signal intensities were monitored along retino-recipient projections in naïve animals. Furthermore, the impact of acute CNS fiber injury on MnCl2 propagation was investigated following optic nerve (ON) crush injury. In rodents, mechanical crush of the ON that causes breakage of axonal cylinders (axonotmesis) has become an established experimental paradigm to study traumatic responses of CNS fiber projections (Benowitz and Yin, 2008). In this paradigm, the sensitivity of MEMRI to identify axonal regrowth was correlated with classical histological procedures 12 months after injury.

Further, MEMRI was addressed on instances of pathologic brain senescence. A variety of neurodegenerative diseases involve apoptotic loss of neuronal cell bodies as well as atrophy and degradation of axonal trajectories such as stroke, Alzheimer's dementia, amyotrophic lateral sclerosis and glaucoma (Ferri et al., 2003, Libby et al., 2005, Stokin et al., 2005). The transcription factor NF-κB controls neuronal maintenance and mice with a deletion of the p50 subunit of NF-κB (p50KO/KO) have been used as an experimental model for age-dependent axonal degeneration in the visual system (Takahashi et al., 2007). To further test MEMRI in the detection of projection-specific fiber atrophy we used NF-κB p50 mutant mice as a model for chronic neuronal and axonal degeneration and compared their visual projection with other pigmented and non-pigmented mouse strains.

To define overall feasibility of MnCl2 application in mice we performed susceptibility and toxicity studies and functional visual tests after both single as well as repetitive tracer applications in individual animals and demonstrate cell type-specific vulnerabilities towards MnCl2 exposure.

In summary, our study extends the already available studies on MEMRI by providing detailed biophysical and pharmacological information on optimal dosages, transport kinetics and toxicity of MnCl2 especially for mice. Toxicity studies were confirmed by histological post mortem and functional in vivo investigations. As a technological advantage, we introduce a MRI protocol making high resolution imaging feasible in small rodents even with a low field 3 T scanner. This protocol proved useful in mouse models of neuroregeneration and Waller degeneration to detect regrowth and atrophy of CNS axons. With respect to the FDA-approved manganese derivative Mangafodipir, we suggest this MEMRI technique to be highly promising for further single and serial in vivo applications, e.g. to serve as a tool for early diagnostics and therapeutic approval strategies in humans.

Section snippets

Animals and injury models

For pharmacokinetic and injury studies inbred C57BL/6 (B6) mice of mixed gender and an average age of 15 weeks were used. To investigate mouse strain specific differences, additional studies were performed using NMRI outbred and Balb/c inbred mice. As a genetic model for chronic neuronal and axonal degeneration (Takahashi et al., 2007), mice lacking the p50 subunit of NF-κB on a B6 background at 10 months of age were used (Sha et al., 1995). Animals were kept under controlled conditions in a

MRI protocol for MEMRI in mice with a 3 T clinical scanner

Accounting for the limited field strength of our clinical 3 T scanner, we established a novel MEMRI protocol with modulated parameters. To improve SNR we repeated each scan twice with internal averaging, and repeated this 10 min examination 3 times for later averaging of the magnitude images. This allowed for an acquisition time of 5 min for one single scan of the entire volume. The 35 min total scan time included 5 min localizer and pre-scans and 30 min for the actual MEMR imaging.

Since a 2D

Discussion

There is increasing demand for high resolution in vivo imaging of neuronal circuits and reasonable visualization of fiber projections within the intact and pathologic brain. This need is reflected by recent technical innovations, such as ultramicroscopy developed by Dodt et al. (2007), which enables experimental illustration of complete neuronal networks inside the mouse brain. The advantage of this method is the superb quality in cellular resolution. However, since it requires tissue fixation

Abbreviations

    ON

    optic nerve

    ONI

    optic nerve injury

    CNR

    contrast-to-noise ratio

    CNS

    central nervous system

    CRALBP

    cellular retinaldehyde-binding protein

    CTX

    cholera toxin

    Cyc/deg

    cycle per degree

    3D

    three-dimensional

    DTI

    diffusion tensor imaging

    DTT

    diffusion tensor tractography

    DWI

    diffusion weighted imaging

    fMRI

    functional MRI

    GCL

    ganglion cell layer

    GFAP

    glial fibrillary acidic protein

    Ivit

    intravitreal injection

    LGN

    lateral geniculate nucleus

    MEMRI

    manganese enhanced MRI

    MIP

    maximum intensity projection

    MRI

    magnetic resonance imaging

    NF-κB

Acknowledgments

We thank I. Krumbein and S. Tausch for technical support.

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    Present address: Georg-August-University, Bernstein Focus Neurotechnology and Johann-Friedrich-Blumenbach Institute for Zoology und Anthropology, Berliner Strasse 28, 37073 Göttingen, Germany.

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