A.K. is supported by the Oppenheim Foundation and R.H. is supported by the Velux Foundation. We thank I. Krumbein for technical and K. Buder for histological support, and J. Goldschmidt (Leibniz Institute for Neurobiology, Magdeburg, Germany) for technical advice on TIMM staining.

All animal interventions are performed in accordance with the European Convention for Animal Care and Use of Laboratory Animals and the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. All experiments are approved by the local ethics committee. The procedure of ON injury in mice is described elsewhere9.
1. Intravitreal Manganese Injection
<ol>
	<li>Perform the Mn2+ injection 24 hr prior to the MR scan with the help of an assistant. Anesthetize the animals by intraperitoneal injection of a 5% chloral hydrate solution (420-450 mg/kg body weight in sterile PBS). For additional topical anesthesia, apply one drop of liquid conjuncain (0.4% oxybuprocaine hydrochloride) to the cornea prior to eye puncture. To inject 15 nmol Mn2+ per eye prepare a 7.5 mM MnCl2 solution, e.g., by diluting 1 L of 1 M MnCl2 stock solution in 132 L H2O. Load 5 &#181;l of the final solution into a 5 &#181;l Hamilton syringe connected to a 34 G&#160;small hub removable needle (RN needle).</li>
	<li>When starting with the right eye, position the mouse left-sided under a binocular microscope and gently open and fix the right eye between the thumb and forefinger of your left hand. Pick up the syringe with your right hand and take hold of the needle close to the tip. For atraumatic puncture of the eye bulb, carefully insert the needle into the vitreous body at the infero-temporal circumference approximately 1 mm distal to the limbus, thereby sparing scleral vessels.</li>
	<li>Next, the assistant slowly applies the total volume of 2 &#956;l&#160;while controlling the scale of the Hamilton syringe. During this procedure, monitor optimal needle placement under the microscope and avoid puncture of the lens or spilling of the liquid. Keep the needle statically inserted for an additional 30 sec, then withdraw it slowly to minimize liquid leakage from the injection site.</li>
	<li>Throughout the procedure, special care should be taken to avoid pressure to the eye. Likewise, avoid harsh or numerous attempts to puncture the eye bulb. Since Mn2+ uptake into RGCs and transport along the ON is already saturated at 15 nmol MnCl2, this minimizes signal variations by slightly imprecise injection volumes. For bilateral signal enhancement of the visual projection, repeat the injection procedure for the left eye.</li>
	<li>Apply ofloxacin-containing (3 mg/ml) eye drops and panthenol-containing ointment once after the procedure to prevent ocular infections and drying of the eye. Return the mice to their cages under normal housing conditions until the start of the MR scan.</li>
</ol>
2. Animal Preparation for MRI
<ol>
	<li>Anesthetize the mouse by administration of a 2%/98% isoflurane/oxygen gas mixture. Mount the mouse on a mouse holder in an almost horizontal, untwisted position. Insert it into the MR coil, which is then adjusted inside the MR scanner. Monitor respiration and heart rate by appropriate systems. For technical details, see Herrmann&#160;et al20.</li>
	<li>During MRI, supply anesthesia by continuous insufflation of an initially 1.5%/98.5% isoflurane/oxygen gas mixture via an evaporator connected to the mouse head holder by an integrated tube. During the scan, adjust the deepness of anesthesia according to the recorded vital parameters (i.e. aim for a stable respiration rate of about 40 breaths per minute). Using a heating device keep the body surface temperature stable between 35 and 37 &#176;C, as measured by a thermal sensor positioned at the abdominal site of the mouse. For technical details, see Herrmann&#160;et al17.</li>
	<li>After the scan, liberate the mouse from the holder and supply with pure oxygen to accelerate recovery from anesthesia. Additionally, keep the body temperature stable by using a red light heating source.</li>
</ol>
3. MRI Protocol
<ol>
	<li>The protocol is validated for a 3 Tesla scanner equipped with a dedicated, SNR-efficient, small animal coil (linearly polarized Litz coil) with an effective field of view of 35 mm &#215; 38 mm diameter. Operate the coil in transmit-receive mode.</li>
	<li>With the animal in its final position, adjust the tune and match of the coil with the aid of a frequency analyzer. Manually adjust the transmitter reference voltage and the shim currents to optimize image homogeneity and quality.</li>
	<li>Acquire T1 weighted 2D TSE images with a resolution of 0.5 mm &#215; 0.5 mm &#215; 2 mm in sagittal and transversal view for planning. Using the planning MR scans, acquire the MEMR images in coronal measurement direction, rotated to be parallel to the animal&#8217;s head with phase encoding along the left-right direction. To minimize acquisition time, use a rectangular field of view adjusted to the actual head dimensions. Employ a spoiled 3D FLASH sequence (VIBE 3D) using the following parameters: base matrix 256, field of view 54 mm &#215; 50.65 mm &#215; 14.08 mm, using 93.8% rectangular field of view in phase encode direction, and 128 slices of 0.11-mm slice thickness with slice resolution set to 61%.</li>
	<li>Activate the in-plane interpolation to create final images with 512 &#215; 480 &#215; 128, providing an effective resolution of 0.21 mm &#215; 0.21 mm &#215; 0.18 mm (0.1 mm &#215; 0.1 mm &#215; 0.09 mm interpolated), echo time TE = 6.51 msec, repetition time TR = 16 msec, bandwidth = 160 Hz/px, flip angle = 22&#176;. Apply two averages and three repetitions to achieve a total acquisition time (TA) of approximately 30 min.</li>
</ol>
4. MRI Data Analysis
<ol>
	<li>Analyze the data using the software syngo fastView. For quantitative signal enhancement, select defined regions of interest in 2D planar MRI recordings and determine signal intensities (SI) of the enhanced structure (SIMEMRI), tissue background (SIbackgr), and the standard deviation of the noise (SDN). Where necessary, use a mouse brain atlas to facilitate neuro-anatomical orientation for LGN and SC structures. Calculate the contrast-to-noise ratio (CNR) using the formula:</li>
	<li>CNR = (SIMEMRI&#8211; SIbackgr) / SDN</li>
	<li>Quantify three consecutive images for mean CNR calculation for each sample. In bilaterally injected animals, analyze each hemisphere independently.</li>
	<li>For depiction of horizontal, coronal and sagittal images, calculate multiplanar reconstructions from the original 3D MRI data set. These processed images are not recommended for quantitative analysis. To create animated 3D reconstructions (maximum intensity projections, MIPs) of the retino-tectal projection, use an angiography post-processing software module.</li>
</ol>
5. Mn2+ Autometallography (TIMM Staining)
<ol>
	<li>For TIMM staining of Mn2+ traced brain structures following MEMRI, inject a dosage of 15-150 nmol Mn2+ ivit 24 hr prior to imaging.</li>
	<li>After the MR scan, perfuse the animals with 30 ml ice-cold 0.325% Na2S in PBS (pH 7.4). Dissect retinae and freeze samples in Frozen section.</li>
	<li>Cut sequential, equatorial sections of 15 &#181;m thickness on a cryotome.</li>
	<li>Perform TIMM staining21 in the absence of fixative and cryoprotection according to Angenstein et al22.</li>
</ol>
6. Statistical Analysis
Perform statistical analyses using the Student&#8217;s t-test for single comparisons, followed by post hoc ANOVA. Data are presented as mean &#177; standard error. Individual N numbers are given separately for each experiment. Results reaching P &#8804; 0.05 are considered statistically significant (P &#8804; 0.05, *; P &#8804; 0.01, **; P &#8804; 0.001, ***).

<ol>
	<li>Kretz, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simvastatin+promotes+heat+shock+protein+27+expression+and+Akt+activation+in+the+rat+retina+and+protects+axotomized+retinal+ganglion+cells+in+vivo.">Simvastatin promotes heat shock protein 27 expression and Akt activation in the rat retina and protects axotomized retinal ganglion cells in vivo.</a> Neurobiol Dis. 21, 421-430 (2006).</li><li>Lima, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Combinatorial+therapy+stimulates+long-distance+regeneration,+target+reinnervation,+and+partial+recovery+of+vision+after+optic+nerve+injury+in+mice.">Combinatorial therapy stimulates long-distance regeneration, target reinnervation, and partial recovery of vision after optic nerve injury in mice.</a> Int Rev Neurobiol. 106, 153-172 (2012).</li><li>Lima, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Full-length+axon+regeneration+in+the+adult+mouse+optic+nerve+and+partial+recovery+of+simple+visual+behaviors.">Full-length axon regeneration in the adult mouse optic nerve and partial recovery of simple visual behaviors.</a> Proc Natl Acad Sci U S A. 109, 9149-9154 (2012).</li><li>Goetze, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vision+and+visual+cortical+maps+in+mice+with+a+photoreceptor+synaptopathy:+reduced+but+robust+visual+capabilities+in+the+absence+of+synaptic+ribbons.">Vision and visual cortical maps in mice with a photoreceptor synaptopathy: reduced but robust visual capabilities in the absence of synaptic ribbons.</a> Neuroimage. 49, 1622-1631 (2010).</li><li>Luo, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three-dimensional+evaluation+of+retinal+ganglion+cell+axon+regeneration+and+pathfinding+in+whole+mouse+tissue+after+injury.">Three-dimensional evaluation of retinal ganglion cell axon regeneration and pathfinding in whole mouse tissue after injury.</a> Exp Neurol. 247, 653-662 (2013).</li><li>Pautler, R. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+neuronal+tract+tracing+using+manganese-enhanced+magnetic+resonance+imaging.">In vivo neuronal tract tracing using manganese-enhanced magnetic resonance imaging.</a> Magn Reson Med. 40, 740-748 (1998).</li><li>Watanabe, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mapping+of+retinal+projections+in+the+living+rat+using+high-resolution+3D+gradient-echo+MRI+with+Mn2+-induced+contrast.">Mapping of retinal projections in the living rat using high-resolution 3D gradient-echo MRI with Mn2+-induced contrast.</a> Magn Reson Med. 46, 424-429 (2001).</li><li>Pautler, R. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo,+trans-synaptic+tract-tracing+utilizing+manganese-enhanced+magnetic+resonance+imaging+(MEMRI).">In vivo, trans-synaptic tract-tracing utilizing manganese-enhanced magnetic resonance imaging (MEMRI).</a> NMR biomed. 17, 595-601 (2004).</li><li>Haenold, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Magnetic+resonance+imaging+of+the+mouse+visual+pathway+for+in+vivo+studies+of+degeneration+and+regeneration+in+the+CNS.">Magnetic resonance imaging of the mouse visual pathway for in vivo studies of degeneration and regeneration in the CNS.</a> Neuroimage. 59, 363-376 (2012).</li><li>Lindsey, J. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Magnetic+resonance+imaging+of+the+visual+system+in+vivo:+transsynaptic+illumination+of+V1+and+V2+visual+cortex.">Magnetic resonance imaging of the visual system in vivo: transsynaptic illumination of V1 and V2 visual cortex.</a> Neuroimage. 34, 1619-1626 (2007).</li><li>Bearer, E. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+neuronal+activity+and+kinesin+on+tract+tracing+by+manganese-enhanced+MRI+(MEMRI).">Role of neuronal activity and kinesin on tract tracing by manganese-enhanced MRI (MEMRI).</a> Neuroimage. 37, S37-S46 (2007).</li><li>Mendonca-Dias, M. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Paramagnetic+contrast+agents+in+nuclear+magnetic+resonance+medical+imaging.">Paramagnetic contrast agents in nuclear magnetic resonance medical imaging.</a> Semin Nucl Med. 13, 364-376 (1983).</li><li>Thuen, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Manganese-enhanced+MRI+of+the+optic+visual+pathway+and+optic+nerve+injury+in+adult+rats.">Manganese-enhanced MRI of the optic visual pathway and optic nerve injury in adult rats.</a> J Magn Reson Imaging. 22, 492-500 (2005).</li><li>Sandvig, I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+MRI+of+olfactory+ensheathing+cell+grafts+and+regenerating+axons+in+transplant+mediated+repair+of+the+adult+rat+optic+nerve.">In vivo MRI of olfactory ensheathing cell grafts and regenerating axons in transplant mediated repair of the adult rat optic nerve.</a> NMR biomed. 25, 620-631 (2012).</li><li>Chan, K. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+retinotopic+mapping+of+superior+colliculus+using+manganese-enhanced+magnetic+resonance+imaging.">In vivo retinotopic mapping of superior colliculus using manganese-enhanced magnetic resonance imaging.</a> Neuroimage. 54, 389-395 (2011).</li><li>Chan, K. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+chromium-enhanced+MRI+of+the+retina.">In vivo chromium-enhanced MRI of the retina.</a> Magn Reson Med. 68, 1202-1210 (2012).</li><li>Herrmann, K. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Possibilities+and+limitations+for+high+resolution+small+animal+MRI+on+a+clinical+whole-body+3T+scanner.">Possibilities and limitations for high resolution small animal MRI on a clinical whole-body 3T scanner.</a> Magma. 25, 233-244 (2012).</li><li>Villegas-Perez, M. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+and+protracted+phases+of+retinal+ganglion+cell+loss+follow+axotomy+in+the+optic+nerve+of+adult+rats.">Rapid and protracted phases of retinal ganglion cell loss follow axotomy in the optic nerve of adult rats.</a> J Neurobiol. 24, 23-36 (1993).</li><li>Takahashi, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+spontaneous+optic+neuropathy+in+NF-&#954;&#914;50-deficient+mice:+requirement+for+NF-&#954;&#914;p50+in+ganglion+cell+survival.">Development of spontaneous optic neuropathy in NF-&#954;&#914;50-deficient mice: requirement for NF-&#954;&#914;p50 in ganglion cell survival.</a> Neuropathol Appl Neurobiol. 33, 692-705 (2007).</li><li>Herrmann, K. H. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MRI+compatible+small+animal+monitoring+and+triggering+system+for+whole+body+scanners.">MRI compatible small animal monitoring and triggering system for whole body scanners.</a> Z Med Phys. 24, 55-64 (2013).</li><li>Danscher, G., Zimmer, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+improved+Timm+sulphide+silver+method+for+light+and+electron+microscopic+localization+of+heavy+metals+in+biological+tissues.">An improved Timm sulphide silver method for light and electron microscopic localization of heavy metals in biological tissues.</a> Histochemistry. 55, 27-40 (1978).</li><li>Angenstein, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Manganese-enhanced+MRI+reveals+structural+and+functional+changes+in+the+cortex+of+Bassoon+mutant+mice.">Manganese-enhanced MRI reveals structural and functional changes in the cortex of Bassoon mutant mice.</a> Cereb cortex. 17, 28-36 (2007).</li><li>Thuen, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Manganese-enhanced+MRI+of+the+rat+visual+pathway:+acute+neural+toxicity,+contrast+enhancement,+axon+resolution,+axonal+transport,+and+clearance+of+Mn(2).">Manganese-enhanced MRI of the rat visual pathway: acute neural toxicity, contrast enhancement, axon resolution, axonal transport, and clearance of Mn(2).</a> J Magn Reson Imaging. 28, 855-865 (2008).</li><li>Lehmann, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vision+and+visual+plasticity+in+ageing+mice.">Vision and visual plasticity in ageing mice.</a> Restor Neurol Neurosci. 30, 161-178 (2012).</li><li>Takeda, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Manganese+transport+in+the+neural+circuit+of+rat+CNS.">Manganese transport in the neural circuit of rat CNS.</a> Brain Res Bull. 45, 149-152 (1998).</li><li>Nairismagi, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Manganese-enhanced+magnetic+resonance+imaging+of+mossy+fiber+plasticity+in+vivo.">Manganese-enhanced magnetic resonance imaging of mossy fiber plasticity in vivo.</a> Neuroimage. 30, 130-135 (2006).</li><li>Smith, K. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+axonal+transport+rates+decrease+in+a+mouse+model+of+Alzheimer's+disease.">In vivo axonal transport rates decrease in a mouse model of Alzheimer's disease.</a> Neuroimage. 35, 1401-1408 (2007).</li><li>Berkowitz, B. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Noninvasive+and+simultaneous+imaging+of+layer-specific+retinal+functional+adaptation+by+manganese-enhanced+MRI.">Noninvasive and simultaneous imaging of layer-specific retinal functional adaptation by manganese-enhanced MRI.</a> Invest Ophthalmol Vis Sci. 47, 2668-2674 (2006).</li><li>Schnapf, J. L. B. D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=How+photoreceptor+cells+respond+to+light.">How photoreceptor cells respond to light.</a> Sci. Am. 256 (8), (1987).</li><li>Yu, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+auditory+brain+mapping+in+mice+with+Mn-enhanced+MRI.">In vivo auditory brain mapping in mice with Mn-enhanced MRI.</a> Nat Neurosci. 8, 961-968 (2005).</li><li>Sun, S. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Noninvasive+topical+loading+for+manganese-enhanced+MRI+of+the+mouse+visual+system.">Noninvasive topical loading for manganese-enhanced MRI of the mouse visual system.</a> Invest Ophthalmol Vis Sci. 52, 3914-3920 (2011).</li><li>Sun, S. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Impact+of+repeated+topical-loaded+manganese-enhanced+MRI+on+the+mouse+visual+system.">Impact of repeated topical-loaded manganese-enhanced MRI on the mouse visual system.</a> Invest Ophthalmol Vis Sci. 53, 4699-4709 (2012).</li></ol>

The authors declare that they have no competing financial interests.

MEMRI of the visual system extends conventional neurobiological techniques for assessing functionality under na&#239;ve and pathological conditions. Apart from providing a unique insight into the integrity of an isolated CNS fiber tract, MEMRI can be easily supplemented with behavioral tests, e.g., optometry and visually based water tasks, to investigate the immediate consequences of a given paradigm for visual perception. It also links electrophysiological and histological investigations with functional visual ...

Based on its favorable neuro-anatomical structure the rodent visual system offers unique possibilities to evaluate pharmacological compounds and their capability to mediate neuroprotection1 or pro-regenerative effects2,3. Moreover, it allows studies on the functional and neuro-anatomical characteristics of mouse mutants, as recently exemplified for mice lacking the presynaptic scaffolding protein Bassoon4. Furthermore, a broad spectrum of supplementary tools affords additional featuring of retinal ganglion cell (RGC) and RGC axon numbers as well as RGC activity, e.g., by electroretinography and behavioral tests, and the determination of cortical rearrangements by optical imaging of intrinsic signals. The latest technical developments in laser microscopy enable the in situ visualization of RGC regeneration by deep tissue fluorescence imaging in whole mount specimens of optic nerve (ON) and brain. In this histological approach, tetrahydrofuran based tissue clearing in combination with light sheet fluorescence microscopy permits the resolution of single fibers that re-enter into the deafferented ON and optic tract 5. While such techniques might be superior in resolution and determination of growth patterns, they do not enable repetitive and longitudinal analyses of individual growth events, which are particularly desired to assess the process of long term regeneration.
Contrast-enhanced MRI has been employed for the minimal invasive visualization of the retino-tectal projection in mice and rats6,7. This can be achieved by direct intraocular delivery of paramagnetic ions (e.g., Mn2+) to retinal cells. As a calcium analog, Mn2+ is incorporated into RGC somata via voltage-gated calcium channels and actively transported along the axonal cytoskeleton of the intact ON and optic tract. While it accumulates in brain nuclei of the visual projection, i.e. the lateral geniculate nucleus (LGN) and superior colliculus (SC), transsynaptic propagation into the primary visual cortex appears negligible8,9, although it may occur10,11. Under MR sequencing, paramagnetic Mn2+ augments MR contrast mainly by shortening T1 spin-lattice relaxation time12. Such Mn2+ enhanced MRI (MEMRI) has been successfully applied in various neuro-anatomical and functional studies of rats, including the assessment of axonal regeneration and degeneration after ON injury13,14, the precise anatomical mapping of the retino-tectal projection15, as well as the determination of axonal transport characteristics after pharmacological treatment16. Recent refinements in the dosage, toxicity, and kinetics of neuronal Mn2+ uptake and transport, as well as improved MRI protocols have extended its application to studies on transgenic mice9 using 3 Tesla scanners commonly used in clinical practice17.
Here, we present a MEMRI protocol suitable for longitudinal in vivo imaging of the mouse retino-tectal projection and exemplify its applicability by assessing Mn2+ dependent signal enhancement under na&#239;ve and various neurodegeneration conditions. Our protocol places specific emphasis on MR data acquisition in a moderate 3 T magnetic field that is generally more accessible than dedicated animal scanners. In na&#239;ve mice, we illustrate how tract-specific signal intensity can be substantially and reproducibly become increased after intravitreal (ivit) Mn2+ application. Quantitatively, Mn2+ propagation along the visual projection occurs independently of the normal aging process (measured between 3 and 26 month old mice) and augmentation is refractory to visual stimulation and adaptation to darkness. In contrast, Mn2+ enrichment in thalamic and midbrain centers is diminished following acute ON crush injury18 as well as in nfkb1 knock-out mice (p50KO) suffering from spontaneous apoptotic RGC death and ON degeneration19. Thus, in expansion to conventional histological analysis, longitudinal MEMRI analysis of individual animals enables profiling of unique kinetics of neurodegenerative processes. This should prove useful for studies on neuroprotection and axonal regeneration associated with pharmacological or genetic interventions.

<table border="0" cellpadding="0" cellspacing="0" width="934">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:363px;">Manganese (II) chloride solution 1M</td>
			<td style="width:269px;">Sigma Aldrich, Taufkirchen, Germany</td>
			<td style="width:119px;">M1787</td>
			<td style="width:184px;">MEMRI contrast reagent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;"></td>
			<td></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Conjuncain</td>
			<td>Dr. Mann Pharma, Berlin, Germany</td>
			<td style="width:119px;">PZN 7617666</td>
			<td>0.4% oxybuprocaine hydrochloride</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Floxal eye drops</td>
			<td>Dr. Mann Pharma, Berlin, Germany</td>
			<td style="width:119px;">PZN 3820927</td>
			<td>3 mg/ml ofloxacin</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ointment panthenol</td>
			<td>Jenapharm, Jena, Germany</td>
			<td style="width:119px;">PZN 3524531</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Chloral hydrate&#160;</td>
			<td style="width:269px;">Sigma Aldrich, Taufkirchen, Germany</td>
			<td style="width:119px;">C8383</td>
			<td>420-450 mg/kg body weight</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Isoflurane</td>
			<td>Actavis, Munich, Germany</td>
			<td style="width:119px;">PZN 7253744</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;"></td>
			<td></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Hamilton syringe&#160;</td>
			<td style="width:269px;">Hamilton Company, Reno, NV, USA</td>
			<td style="width:119px;">7634-01</td>
			<td>SYR 5 &#181;l, 75 RN, no NDL</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">34 G&#160; needle (34/35/pst4/tapN)</td>
			<td style="width:269px;">Hamilton Company, Reno, NV, USA</td>
			<td style="width:119px;">207434/00</td>
			<td>removable needle RN, 34 gauge, lenght 38.1 mm, point style 4</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Binocular Stemi-2000</td>
			<td style="width:269px;">Zeiss, Oberkochen, Germany</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:363px;"></td>
			<td style="width:269px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">3T MRI scanner Magnetom TIM Trio</td>
			<td colspan="2">Siemens Medical Solutions, Erlangen, Germany</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Rat head coil</td>
			<td>Doty Scientific Inc., Columbia, SC, USA</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Mouse holder</td>
			<td style="width:269px;"></td>
			<td style="width:119px;"></td>
			<td>custom made</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Red light lamp</td>
			<td style="width:269px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;"></td>
			<td style="width:269px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:363px;">Frozen section medium NEG-50</td>
			<td style="width:269px;">Thermo Fisher Scientific, Schwerte, Germany</td>
			<td style="width:119px;">6502</td>
			<td>tissue embedding for cryo-sections</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;"></td>
			<td style="width:269px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:363px;">Sodium dihydrogen phosphate monohydrate (NaH2PO4)</td>
			<td>Merck, Darmstadt, Germany</td>
			<td style="width:119px;">106346</td>
			<td rowspan="2" style="width:184px;">for sulfide perfusion&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:363px;">Sodium sulfide nonahydrate (Na2S &#215; 9 H2O)</td>
			<td>Sigma Aldrich, Taufkirchen, Germany</td>
			<td style="width:119px;">208043</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:363px;">gum arabic</td>
			<td style="width:269px;">Roth, Arlesheim, Switzerland</td>
			<td style="width:119px;">4159</td>
			<td rowspan="5">for TIMM staining</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:363px;">Hydroquinone (C6H6O2)</td>
			<td style="width:269px;">Roth, Arlesheim, Switzerland</td>
			<td style="width:119px;">3586</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:363px;">Citric acid (C6H8O7)</td>
			<td style="width:269px;">Roth, Arlesheim, Switzerland</td>
			<td style="width:119px;">6490</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:363px;">Tri-sodium citrate dihydrate (C6H5Na3O7 x 2H2O)</td>
			<td style="width:269px;">Merck, Darmstadt, Germany</td>
			<td style="width:119px;">106448</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:363px;">Silver nitrate (AgNO3)</td>
			<td style="width:269px;">Roth, Arlesheim, Switzerland</td>
			<td style="width:119px;">7908</td>
		</tr>
	</tbody>
</table>

in vivo imaging of optic nerve fiber integrity by contrast-enhanced mri in mice

The rodent visual system encompasses retinal ganglion cells and their axons that form the optic nerve to enter thalamic and midbrain centers, and postsynaptic projections to the visual cortex. Based on its distinct anatomical structure and convenient accessibility, it has become the favored structure for studies on neuronal survival, axonal regeneration, and synaptic plasticity. Recent advancements in MR imaging have enabled the in vivo visualization of the retino-tectal part of this projection using manganese mediated contrast enhancement (MEMRI). Here, we present a MEMRI protocol for illustration of the visual projection in mice, by which resolutions of (200 &#181;m)3 can be achieved using common 3 Tesla scanners. We demonstrate how intravitreal injection of a single dosage of 15 nmol MnCl2 leads to a saturated enhancement of the intact projection within 24 hr. With exception of the retina, changes in signal intensity are independent of coincided visual stimulation or physiological aging. We further apply this technique to longitudinally monitor axonal degeneration in response to acute optic nerve injury, a paradigm by which Mn2+ transport completely arrests at the lesion site. Conversely, active Mn2+ transport is quantitatively proportionate to the viability, number, and electrical activity of axon fibers. For such an analysis, we exemplify Mn2+ transport kinetics along the visual path in a transgenic mouse model (NF-&#954;B p50KO) displaying spontaneous atrophy of sensory, including visual, projections. In these mice, MEMRI indicates reduced but not delayed Mn2+ transport as compared to wild type mice, thus revealing signs of structural and/or functional impairments by NF-&#954;B mutations.
In summary, MEMRI conveniently bridges in vivo assays and post mortem histology for the characterization of nerve fiber integrity and activity. It is highly useful for longitudinal studies on axonal degeneration and regeneration, and investigations of mutant mice for genuine or inducible phenotypes.

The ability of this imaging technique to accurately assess the vitality and functionality of the visual projection relies upon precise application of a nontoxic Mn2+ dosage to the vitreous body and its uptake by RGCs. This major assumption is tested in Figure 1, where layer specific Mn2+ uptake is demonstrated by autometallography (TIMM staining)21. Retina sections were analyzed at 24 hr after ivit application of either 15 nmol or 150 nmol Mn2+, or PBS...

Watch this Scientific Journal Video about In vivo Imaging of Optic Nerve Fiber Integrity by Contrast-Enhanced MRI in Mice at JoVE.com

In vivo Imaging of Optic Nerve Fiber Integrity by Contrast-Enhanced MRI in Mice

This video illustrates a method, using a clinical 3 T scanner, for contrast-enhanced MR imaging of the na&#239;ve mouse visual projection and for repetitive and longitudinal in vivo studies of optic nerve degeneration associated with acute optic nerve crush injury and chronic optic nerve degeneration in knock-out mice (p50KO).

In vivo Imaging of Optic Nerve Fiber Integrity by Contrast-Enhanced MRI in Mice

The rodent visual system encompasses retinal ganglion cells and their axons that form the optic nerve to enter thalamic and midbrain centers, and ...

in-vivo-imaging-optic-nerve-fiber-integrity-contrast-enhanced-mri

Research

JoVE Journal

Neuroscience

13.2K Views.  Jena University Hospital. This video illustrates a method, using a clinical 3 T scanner, for contrast-enhanced MR imaging of the naïve mouse visual projection and for repetitive and longitudinal in vivo studies of optic nerve degeneration associated with acute optic nerve crush injury and chronic optic nerve degeneration in knock-out mice (p50KO).