Name: in vivo imaging of optic nerve fiber integrity by contrast-enhanced mri in mice
Uploaded: 2014-07-22T14:00:00
Description: Watch this Scientific Journal Video about In vivo Imaging of Optic Nerve Fiber Integrity by Contrast-Enhanced MRI in Mice at JoVE.com

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 assista...

<ol>
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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>
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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 ...

Research

JoVE Journal

Neuroscience

Methods for Experimental Manipulations after Optic Nerve Transection in the Mammalian CNS

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