Manipulation of tissue contrast using contrast agents for enhanced MR microscopy in ex vivo mouse brain

Abstract

Detailed 3D mouse brain images may promote better understanding of phenotypical differences between normal and transgenic/mutant mouse models. Previously, a number of magnetic resonance microscopy (MRM) studies have successfully established brain atlases, revealing genotypic traits of several commonly used mouse strains. In such studies, MR contrast agents, mainly gadolinium (Gd) based, were often used to reduce acquisition time and improve signal-to-noise ratio (SNR). In this paper, we intended to extend the utility of contrast agents for MRM applications. Using Gd-DTPA and MnCl(2), we exploited the potential use of MR contrast agents to manipulate image contrast by drawing upon the multiple relaxation mechanisms and tissue-dependent staining properties characteristic of each contrast agent. We quantified r(1) and r(2) of Gd-DTPA and MnCl(2) in both aqueous solution and brain tissue and demonstrated the presence of divergent relaxation mechanisms between solution and tissue for each contrast agent. Further analyses using nuclear magnetic resonance dispersion (NMRD) of Mn(2+) in ex vivo tissue strongly suggested macromolecule binding of Mn(2+), leading to increased T(1) relaxation. Moreover, inductively coupled plasma (ICP) mass spectroscopy revealed that MnCl(2) had higher tissue affinity than Gd-DTPA. As a result, multiple regions of the brain stained by the two agents exhibited different image contrasts. Our results show that differential MRM staining can be achieved using multiple MR contrast agents, revealing detailed cytoarchitecture, and may ultimately offer a window for investigating new techniques by which to understand biophysical MR relaxation mechanisms and perhaps to visualize tissue anomalies even at the molecular level.

Figure 1

CNR vs. TE of mouse brains stained with Gd-DTPA or MnCl₂ at different concentrations based on 2D MGE sequence. Top panel: brains in 0.12, 0.24, or 0.36 mM MnCl₂ showing image contrast between cortex and thalamus on the left, cortex and striatum on the right; bottom panel: brains in 1, 2.5, 5, or 7.5 mM Gd-DTPA, left: cortex v.s. thalamus, right: cortex v.s. striatum.

Figure 2

Changes in tissue T₁ relaxation time over time for mouse brains stained with 5 mM Gd- DTPA and 0.24 mM MnCl₂. These data demonstrate that at least three days are needed for the contrast agent to fully penetrate brain tissue. To ensure good tissue staining, approximately five days are needed before imaging.

Figure 3

Coronal sections of mouse brains stained with 5 mM Gd-DTPA (3A) or 0.24 mM MnCl₂ (3B). Images were acquired using 3D FLASH sequence with different flip angles (from left to right, 10°, 25°, 55°, 70°, and 90°) and TEs (top: TE = 4 ms, bottom: TE = 8 ms).

Figure 4

high-resolution MRM of ex vivo mouse brain stained with 0.24 mM MnCl₂ (4A) or 5 mM Gd- DTPA (4B) reveals detailed anatomical structure with different image contrast in certain regions brain, e.g., cerebellum, cortex, and hippocampus.

Figure 5

Cross-section images of an ex vivo mouse brains stained with 5 mM Gd-DTPA (5A) or 0.36 mM MnCl₂ (5C) and segmented brain structures (5B). These two contrast agents showed different tissue differentiation abilities. For example, Frimbria is better visualized in brain stained with Mn²⁺. Lamina structure of hippocampus is more pronounced, especially the axial view (8C), in brain stained with Mn²⁺. More interestingly, the image contrast of cerebellar cortical layers (granular and molecular layers) is reversed between the brains stained Gd-DTPA and Mn²⁺. By cross examination of MRM images obtained using these two staining agents, detailed anatomical structures can be better resolved.

Figure 6

Changes in R₁, and R₂* values in cerebellar molecular and granular layers of mouse brain after stained with 0.36 mM MnCl₂. The higher ΔR₁ and ΔR₂* in granular layer indicate that Mn²⁺ is possibly accumulated in neuronal cells in this layer.

Figure 7

Images of ex vivo mouse brains fixed without (A) and with (B) Mn²⁺ in 4% PFA during transcardial perfusion. Image contrast is not significantly affected by the different staining protocols.

Figure 8

Regions in the cerebellum of brains stained with MnCl₂ or Gd-DTPA showed correlation with gene expression patterns of Ca²⁺ binding protein calretinin (calb2) in cerebellum and lectithin cholesterol acyltransferase (Lcat). Calb2 and Lcat expression images were obtained from online Allen Brain Atlas (http://www.brain-map.org).