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 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 rats^6,7. 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 negligible^8,9, although it may occur^10,11. 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 injury^13,14, 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 naïve 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 naïve 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 (p50^KO) 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.

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 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 µl of the final solution into a 5 µl Hamilton syringe connected to a 34 G small hub removable needle (RN needle).
2. 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.
3. Next, the assistant slowly applies the total volume of 2 μl 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.
4. 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.
5. 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.

2. Animal Preparation for MRI

1. 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 et al²⁰.
2. 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 °C, as measured by a thermal sensor positioned at the abdominal site of the mouse. For technical details, see Herrmann et al¹⁷.
3. 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.

3. MRI Protocol

1. 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 × 38 mm diameter. Operate the coil in transmit-receive mode.
2. 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.
3. Acquire T₁ weighted 2D TSE images with a resolution of 0.5 mm × 0.5 mm × 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’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 × 50.65 mm × 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%.
4. Activate the in-plane interpolation to create final images with 512 × 480 × 128, providing an effective resolution of 0.21 mm × 0.21 mm × 0.18 mm (0.1 mm × 0.1 mm × 0.09 mm interpolated), echo time T_E = 6.51 msec, repetition time T_R = 16 msec, bandwidth = 160 Hz/px, flip angle = 22°. Apply two averages and three repetitions to achieve a total acquisition time (T_A) of approximately 30 min.

4. MRI Data Analysis

1. 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 (SI_MEMRI), tissue background (SI_backgr), and the standard deviation of the noise (SD_N). 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:
2. CNR = (SI_MEMRI– SI_backgr) / SD_N
3. Quantify three consecutive images for mean CNR calculation for each sample. In bilaterally injected animals, analyze each hemisphere independently.
4. 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.

5. Mn²⁺ Autometallography (TIMM Staining)

1. For TIMM staining of Mn²⁺ traced brain structures following MEMRI, inject a dosage of 15-150 nmol Mn²⁺ ivit 24 hr prior to imaging.
2. 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.
3. Cut sequential, equatorial sections of 15 µm thickness on a cryotome.
4. Perform TIMM staining²¹ in the absence of fixative and cryoprotection according to Angenstein et al²².

6. Statistical Analysis

Perform statistical analyses using the Student’s t-test for single comparisons, followed by post hoc ANOVA. Data are presented as mean ± standard error. Individual N numbers are given separately for each experiment. Results reaching P ≤ 0.05 are considered statistically significant (P ≤ 0.05, *; P ≤ 0.01, **; P ≤ 0.001, ***).

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

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