Introduction

In the rodent’s lissencephalic brain, manganese-enhanced MRI (MEMRI) demonstrated to be an unavoidable technique for the delineation of cytoarchitectural features at unprecedented spatial resolutions in-vivo (1).One of the main drawbacks of manganese ions (Mn²⁺), is their toxicity especially at high doses. To observe anatomical and functional information without artefacts and at high spatial resolutions, ex-vivo MEMRI after in-vivo Mn²⁺ administration have been privileged but only a few studies have been conducted (2).In particular, difficulties to maintain the Mn²⁺ contrast in fixed tissues have been reported (3). Here, an ex-vivo MEMRI quqlitative exploration of the lambs’ brain microstructure at 3T was performed. Perfused lamb brains were soaked in highly concentrated MnCl2 (> 200 mM) baths and Mn²⁺ ions were absorbed by brain tissues. The lamb brains were then imaged using T1-weighted techniques.

Materials and Methods

All experiments were performed in 4 Ile-de-France (IF) female lambs (4 months old, 30-35kg). Each animal was fasted 24-hours prior to intubation. After immobilization, the lamb was intubated after intravenous administration of a mixture of ketamine and xylazine (20 mg/kg). Each lamb was transported to the MRI room, installed prone on the MRI bed and anaesthesia was immediately switched to 1% ISO in medical air through a respirator (Aestiva, GE Healthcare, Datex-Ohmeda, USA). The respirator allowed continuous control of respiration rates. An oximeter was attached to one of the hind-paws allowing for the control of the partial pressure of oxygen and heart rate. The temperature was controlled through MRI-compatible rectal probe. The duration of each MRI session was limited to 150 minutes for each animal. A continuous physiological follow up of the animal status by a veterinary was performed during experiments. All the lambs were euthanized with an overdose of sodium pentobarbitone (25 mg/kg; Merial, Lyon, France) preceded by an injection of heparin. After euthanasia, animals were decapitated and the heads were perfused through both carotid arteries with 2 L sodium nitrite (1%) in NaCl (0.9%) followed by 4 L of cold paraformaldehyde (4%) in 0.1 M phosphate buffer, pH 7.4. The whole brains were collected. After a 48-hour period of postfixation, each brain was soaked in 20% sucrose for cryoprotection. A bulk solution of manganese chloride (MnCl₂) in saline (0.9 %) (200mM, Sigma-Aldrich, St. Louis, MO, USA) was prepared. Three brains were immersed in MnCl₂ for one month and returned to a sucrose bath and conserved at 4° C. MRI was conducted 24 hours and after 3 months after MnCl₂ immersion. During acquisitions, lamb brains were immersed in a water bath at room temperature. In-vivo and ex-vivo MR imaging were conducted on a 3T whole body MR Scanner (Siemens Verio, Erlangen, Germany). A large flex coil surrounding the entire head for reception and the body coil for transmission were used for in-vivo measurements. For ex-vivo measurements, the 12-element head volume coil was used for transmission. MPRAGE images were acquired in-vivo and ex-vivo with parameters (TR/TE/TI=2500/3.18/900 ms ; Flip angle = 12°; NEX= 2 ; FOV=192 x 192 mm2, Matrix size = 384 x 384 x 256; In-vivo Voxel size = 0.5 x 0.5 x 0.5 mm³ ; Ex-vivo Voxel size = 0.5 x 0.5 x 0.2 mm³). ImageJ was used to visualize and investigate images qualitatively.

Results

Without MnCl₂, no contrast was seen between different structures of the ex-vivo lamb brain (Fig 1A) whereas ex-vivo brains soaked in MnCl₂ for 24 hours (Fig.1B) and in-vivo brains (Fig.1C) revealed important differences. In cortical areas, white matter (WM) to gray matter (GM) contrast was seen in-vivo (Fig 2A) including the stria of Gennari (white arrows). After 24-hours in MnCl₂, CSF was replaced by MnCl2-doped liquid (Fig.2B, arrows). After 3 months in sucrose, Mn²⁺ had diffused in the lamb brains. In cortical areas (Fig 2C), WM appeared as dark and other cell layers displayed gray to white contrast (blue arrows). Within 24 hours, lamb brains soaked in MnCl₂ displayed hippocampal layers (Fig.3A and inset) and granulated features in the globus pallidus (Fig.3B and inset) similar to these seen in fiber stained specimens of the sheep brain atlas (Fig.3C) (4). In sagittal and coronal brain slices, 3 months post-immersion, Mn²⁺ uptake was seen in the hippocampi (white boxes) where different microstructures could correspond to CA3 cells and the dentate gyrus (Fig 4 and Fig.5) by reference to the sheep brain atlas (4). Mn2+ also stained cerebellar structures (yellow boxes, Fig 4 and 5) as well as other structures such as the edges of the thalamus (Th), the hypothalamus and the geniculate nucleus (yellow arrows Fig 4 and 5).

Discussion

Despite important susceptibility artefacts, 3D T1-weighted images of ex-vivo lamb brains soaked in a bulk solution of highly concentrated MnCl₂ displayed microstructural features of interest such as laminar structures in the hippocampus and cortical layers as soon as 24 hours post-staining and up to 3 months after staining. Quantitative assessments, which will validate these early findings will be performed.

Conclusion

A novel methodology for ex-vivo MEMRI observation several months after initial staining was proposed. Although only qualitative for the time being, we expect that this work will open new perspectives for MEMRI in gyrencephalic brains.

Acknowledgements

The author acknowledges the help of Martine Batailler and JeaPhilippe Dubois of INRAE Nouzilly, France.

References

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