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

Summary

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 (p50^KO).

Abstract

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 µm)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-κB p50^KO) 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-κB 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.

Introduction

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 effects^2,3. 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 determ....

Protocol

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

1. 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 t....

Results

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.......

Discussion

MEMRI of the visual system extends conventional neurobiological techniques for assessing functionality under naïve 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 .......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Manganese (II) chloride solution 1 M	Sigma Aldrich, Taufkirchen, Germany	M1787	MEMRI contrast reagent
Conjuncain	Dr. Mann Pharma, Berlin, Germany	PZN 7617666	0.4% oxybuprocaine hydrochloride
Floxal eye drops	Dr. Mann Pharma, Berlin, Germany	PZN 3820927	3 mg/ml ofloxacin
Ointment panthenol	Jenapharm, Jena, Germany	PZN 3524531
Chloral hydrate	Sigma Aldrich, Taufkirchen, Germany	C8383	420-450 mg/kg body weight
Hamilton syringe	Hamilton Company, Reno, NV, USA	7634-01	SYR 5 µl, 75 RN, no NDL
34 G needle (34/35/pst4/tapN)	Hamilton Company, Reno, NV, USA	207434/00	removable needle RN, 34 G, length 38.1 mm, point style 4
Binocular Stemi-2000	Zeiss, Oberkochen, Germany
3 T MRI scanner Magnetom TIM Trio	Siemens Medical Solutions, Erlangen, Germany
Rat head coil	Doty Scientific Inc., Columbia, SC, USA
Mouse holder			custom made
Red light lamp
Frozen section medium NEG-50	Thermo Fisher Scientific, Schwerte, Germany	6502	tissue embedding for cryo-sections
Sodium dihydrogen phosphate monohydrate (NaH2PO4·H2O)	Merck, Darmstadt, Germany	106346	for sulfide perfusion
Sodium sulfide nonahydrate (Na2S·9H2O)	Sigma Aldrich, Taufkirchen, Germany	208043
Gum arabic	Roth, Arlesheim, Switzerland	4159	for TIMM staining
Hydroquinone (C6H6O2)	Roth, Arlesheim, Switzerland	3586
Citric acid (C6H8O7)	Roth, Arlesheim, Switzerland	6490
Tri-sodium citrate dihydrate (C6H5Na3O7·2H2O)	Merck, Darmstadt, Germany	106448
Silver nitrate (AgNO3)	Roth, Arlesheim, Switzerland	7908