Elsevier

NeuroImage

Volume 47, Issue 4, 1 October 2009, Pages 1312-1318
NeuroImage

Measurement of cerebral blood volume in mouse brain regions using micro-computed tomography

https://doi.org/10.1016/j.neuroimage.2009.03.083Get rights and content

Abstract

Micro-computed tomography (micro-CT) is an X-ray imaging technique that can produce detailed 3D images of cerebral vasculature. This paper describes the development of a novel method for using micro-CT to measure cerebral blood volume (CBV) in the mouse brain. As an application of the methodology, we test the hypotheses that differences in CBV exist over anatomical brain regions and that high energy demanding primary sensory regions of the cortex have locally elevated CBV, which may reflect a vascular specialization. CBV was measured as the percentage of tissue space occupied by a radio-opaque silicon rubber that fills the vasculature. To ensure accuracy of the CBV measurements, several innovative refinements were made to standard micro-CT specimen preparation and analysis procedures. Key features of the described method are vascular perfusion under controlled pressure, registration of the micro-CT images to an MRI anatomical brain atlas and re-scaling of micro-CT intensities to CBV units with selectable exclusion of major vessels. Histological validation of the vascular perfusion showed that the average percentage of vessels filled was 93 ± 3%. Comparison of thirteen brain regions in nine mice revealed significant differences in CBV between regions (p  < 0.0001) while cortical maps showed that primary visual and auditory areas have higher CBV than primary somatosensory areas.

Introduction

Micro-computed tomography (micro-CT) can provide detailed 3D images of the mouse vascular architecture (Ritman, 2004). Recent applications of micro-CT to the mouse cerebral circulation include the systematic classification of major vessels (Dorr et al., 2007), the detection of atherosclerotic lesions around the circle of Willis (Langheinrich et al., 2007), and the co-registration of capillary-level views of the circulation with the macroscopic vasculature (Heinzer et al., 2006). The use of this technique in the mouse is motivated by a desire to better understand mouse models of human diseases.

This paper describes the application of micro-CT to measure cerebral blood volume (CBV) for characterizing total vascularity in 3D regions of the mouse brain. CBV is defined as the total volume of blood in a given unit volume of brain (Toga and Mazziotta, 2002). Measurement of CBV in local regions of the mouse brain is of particular interest for delineating the phenotypes of models of neurodegenerative diseases that alter cerebral vasculature, such as Alzheimer's disease (Buee et al., 1997).

Before the advent of suitable 3D imaging technologies, the CBV of the whole mouse brain was measured by detecting intravascular radionuclides (Edvinsson et al., 1973). More recently, regional values of CBV were obtained using magnetic resonance imaging (MRI) (Wu et al., 2003); however, resolution was limited to 0.1 mm × 0.1 mm × 0.6 mm due to the time constraints of in vivo MRI scanning. Another technique that allows a higher resolution for CBV mapping is multi-photon laser scanning microscopy (Verant et al., 2007); however, available optics and depth of light penetration limit this technique to the superficial 0.6 mm of cortex over small fields of view. Micro-CT measurement of CBV provides both high-resolution and whole brain coverage for characterizing 3D regions.

In this paper, we present a methodology by which CBV can be measured as the percentage of a volume of tissue occupied by a perfused radio-opaque silicon rubber that remains intravascular. We utilize the principle that for a voxel filled with two components, tissue and radio-opaque contrast agent, the micro-CT image intensity is a weighted average of the attenuation coefficients of each component (Goodenough et al., 1986). Thus, the micro-CT image intensity is linearly related to the proportion of a voxel's volume that is occupied by radio-opaque contrast agent.

The procedure outlined in this paper involves several innovative refinements to standard micro-CT specimen preparation and analysis. First, to permit reproducible measurement of CBV, radio-opaque vascular casts were prepared under controlled pressure. Second, to permit regional comparisons, micro-CT images were registered to an MRI anatomical brain atlas. Third, to better reflect the contribution of local microvessels to CBV, major vessels were excluded from the analysis.

We also address the hypotheses that differences in CBV exist over anatomical brain regions and that highly active primary sensory cortical areas have a particularly rich vascularization to meet their high metabolic demands (Harrison et al., 2002, Harrison, 2006). Specifically, we examine the possibility that primary sensory cortex has a relatively high CBV in the non-stimulated condition, reflecting more dense patterns of vascularization.

Section snippets

Materials and methods

The steps to measure CBV in regions of the mouse brain were: (1) the cerebral vasculature was filled with Microfil (Flow Tech, Inc., Carver, MA, USA), a radio-opaque silicone rubber containing particulate lead chromate and lead sulfate and known for minimal shrinkage (Cortell, 1969); (2) micro-CT images were acquired; (3) micro-CT images were re-scaled to CBV units; (4) CBV images were co-registered to an MRI anatomical brain atlas; (5) CBV was measured over brain regions; (6) small vessel CBV

Results

A representative example of one of the Micro-CT images is shown in three maximum intensity projection views in Fig. 1. All major arteries branching from the circle of Willis and major returning veins and sinuses were identifiable, such as the middle cerebral artery (MCA), the anterior cerebral artery (ACA) and the superior sagittal sinus (SSS) (marked with arrows in Fig. 1). Inspection of the images provided no evidence that Microfil had leaked outside of vessels.

Histological analysis revealed

Discussion

This paper demonstrates the use of micro-CT to generate CBV maps of the mouse, an application that is of particular interest for characterizing cerebral vascular disease phenotypes. Several innovative refinements to standard micro-CT specimen preparation and analysis procedures were developed to meet this objective. To minimize variation due to vessel inflation, we perfused the vascular network at a specified pressure by controlling inflow and outflow of the contrast agent. To permit regional

Disclosure/conflict of interest

We have no conflict of interest.

Acknowledgments

We would like to thank Marvin Estrada of the Lab Animal Services at the Hospital for Sick Children for valuable advice in developing the surgical procedure. We would also like to thank Professor Bojana Stefanovic of the Sunnybrook Health Sciences Centre for her helpful suggestions.

Funding for this research was provided by the Canadian Institutes of Health Research with Funding Reference Number 86734.

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