Magnetic resonance imaging of the mouse visual pathway for in vivo studies of degeneration and regeneration in the CNS
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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In vivo MRI evaluation of anterograde manganese transport along the visual pathway following whole eye transplantation
2022, Journal of Neuroscience MethodsCitation Excerpt :In the future, it may be useful to combine our in vivo MEMRI imaging model system with other modalities for more comprehensive evaluation of the WET outcomes in a single setting. These modalities may include gadolinium-enhanced MRI of aqueous humor dynamics and blood-ocular barriers, optical coherence tomography of retinal morphology and physiology, diffusion tensor/kurtosis MRI of axonal and glial integrity, electrophysiology of retinal, optic nerve, and visual cortical functions by pattern or flash electroretinography, compound action potential recording, and visually evoked potentials (Spees et al., 2018; Wang et al., 2012), blood-oxygenation-level-dependent functional MRI of hemodynamic visual brain responses, behavioral assessments of optokinetic and optomotor reflexes, histological confirmation of in vivo imaging findings at end time points, and their inter-relationships thereof (Haenold et al., 2012; Ho et al., 2015). It is essential to continue developing this WET animal model and the combined in vivo imaging model system to guide clinical translation and to improve the functionality of outcomes in patients (Bourne et al., 2017; Davidson et al., 2016a, 2016b).
Experience-dependent structural plasticity in the adult brain: How the learning brain grows
2021, NeuroImageCitation Excerpt :Daily improvement rates of VA and CS (δVA, δCS) were calculated between days where optometry was performed immediately consecutive without gaps. Brain MRI was performed using a clinical 3T whole body scanner (Magnetom TIM Trio, Siemens Medical Solutions, Erlangen, Germany) equipped with a dedicated rat head volume resonator using a linearly polarized Litz design (Doty Scientific Inc., Columbia, SC, USA) (Haenold et al., 2012; Herrmann et al., 2012). Freely breathing animals were anesthetized by isoflurane (1.7% in oxygen, 1.5 l/min).
Radiation-induced impairment of optic nerve axonal transport in tree shrews and rats monitored by longitudinal manganese-enhanced MRI
2020, NeuroToxicologyCitation Excerpt :Because this study is a longitudinal MEMRI observation, multiple injections of Mn2+ were administered, which may have also affected the axonal transport function. In the literature, the main toxicity effect of Mn2+ is to reduce the number of RGCs (Haenold et al., 2012a; Sun et al., 2012; Thuen et al., 2008). No significant decrease in RGCs was observed in either the irradiation group or the control group in our study.
Manganese-enhanced MR imaging (MEMRI) combined with electrophysiology in the study of cross-modal plasticity in binocularly blind rats
2017, International Journal of Developmental NeuroscienceThe expression of syntaphilin is down-regulated in the optic nerve after axonal injury
2014, Experimental Eye ResearchCitation Excerpt :Recently, deficits in axonal transport have been reported in rat and mouse glaucoma models (Salinas-Navarro et al., 2010; Chidlow et al., 2011) and in human high-pressure secondary glaucoma (Knox et al., 2007). Other optic neuropathies causing RGC death are also characterized by early breakdown of the axonal transport system (Crish et al., 2010; Haenold et al., 2012). A characteristic feature of neurons is the axon, which can be several orders of magnitude longer than the cell body.
Axonal transport rate decreased at the onset of optic neuritis in EAE mice
2014, NeuroImageCitation Excerpt :Intravitreal injection of MnCl2 is a common practice for MEMRI studies. The toxicity of intravitreal injection of MnCl2 in mice and rats has been investigated previously (Bearer et al., 2007; Haenold et al., 2012; Luo et al., 2012; Thuen et al., 2008). In a preliminary study (Lin et al., 2014), we have concluded that intravitreal injection (0.25 μL of 0.2 M MnCl2) slightly affected visual acuity with full recovery a day later without causing axonal injury or loss in C57BL/6 mice.
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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.