MRI of cellular layers in mouse brain in vivo

Abstract

Noninvasive imaging of the brain of animal models demands the detection of increasingly smaller structures by in vivo MRI. The purpose of this work was to elucidate the spatial resolution and structural contrast that can be obtained for studying the brain of C57BL/6J mice by optimized T2-weighted fast spin-echo MRI at 9.4 T. As a prerequisite for high-resolution imaging in vivo, motion artifacts were abolished by combining volatile anesthetics and positive pressure ventilation with a specially designed animal bed for fixation. Multiple substructures in the cortex, olfactory bulb, hippocampus, and cerebellum were resolved at 30 to 40 μm in-plane resolution and 200 to 300 μm section thickness as well as for relatively long echo times of 65 to 82 ms. In particular, the approach resulted in the differentiation of up to five cortical layers. In the olfactory bulb the images unraveled the mitral cell layer which has a thickness of mostly single cells. In the hippocampus at least five substructures could be separated. The molecular layer, Purkinje layer, and granular layer of the cerebellum could be clearly differentiated from the white matter. In conclusion, even without the use of a contrast agent, suitable adjustments of a widely available T2-weighted MRI sequence at high field allow for structural MRI of living mice at near single-cell layer resolution.

Introduction

In recent years MRI has developed into an indispensable research tool for noninvasive imaging of the brain. Complementing histological studies, in vivo MRI offers the detection of more global changes in intact animals but does not suffer from shrinking or other preparation artifacts and allows for a sequential follow-up of the same animal. In addition, because most MRI techniques can be performed in both humans and experimental animals, the method provides a missing link in translational research. In this context, the successful use of (genetically modified) mouse models enhances the demand for detecting increasingly smaller brain structures by in vivo MRI. In terms of absolute spatial resolution this corresponds to isotropic image voxel dimensions of less than 100 μm or voxel sizes of less than 1 nl. In addition, the discrimination of specific structures requires sufficient contrast, or more precisely a certain contrast-to-noise ratio (CNR) that also takes into account the signal-to-noise ratio (SNR) and available measurement time. In fact, because a reduction of the voxel volume in MRI results in a linear decrease of the SNR, the reduced partial volume effect and better contrast between adjacent structures at high resolution might be compromised by a lower SNR. Similarly, a better tissue contrast based on a more pronounced relaxation time weighting is also at the expense of SNR due to more pronounced T1 saturation or T2 attenuation.

Recently, several attempts have been made for high-resolution imaging of the cortical anatomy in humans (Barbier et al., 2002, Walters et al., 2003) advancing the spatial resolution to 240 × 240 × 1000 μm³ (Duyn et al., 2007). In mouse brain, however, high-resolution MRI at 20 to 60 μm isotropic resolution (corresponding to voxel sizes of 8 pl to 0.2 nl) has so far only been achieved ex vivo using sacrificed animals (Badea et al., 2007, Benveniste and Blackband, 2002, Kovacevic et al., 2005). Although of basic value in closely linking MRI to histological assessments, such studies do not fully exploit the specific advantages of in vivo MRI over conventional histology. Nevertheless, only few MRI studies attempted to visualize cortical substructures in living mice (Angenstein et al., 2007, Chahboune et al., 2007, Lee et al., 2005, MacKenzie-Graham et al., 2004). A first challenge stems from movements due to spontaneous breathing (Ma et al., 2008) which bears the risk of impairing or even precluding access to microscopic resolution. Continuous endotracheal intubation and positive pressure ventilation may help to overcome this problem (Brown et al., 1999) and has been shown to allow for repeated measurements of mice, each with a total duration of several hours (Merkler et al., 2005, Boretius et al., 2009). A second difficulty stems from the relatively small amount of white matter (Zhang and Sejnowski, 2000). In humans, the many strongly myelinated structures are particularly useful for mapping the brain into anatomically and functionally distinct areas. For example, in the striate cortex T1-weighted MRI at high spatial resolution reveals the stria of Gennari (Barbier et al., 2002, Clark et al., 1992).

The aim of this study was to explore the practical limits of in vivo MRI of mouse brain with respect to resolution and (native) contrast that can be achieved by suitable MRI sequences on commercially available high-field MRI systems and within reasonable measuring times. These techniques were exploited for the detection of cerebral layers in normal mice, where we were able to achieve near single-cell layer resolution in several brain structures.