Measurement of cerebral blood volume in mouse brain regions using micro-computed tomography
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.
References (33)
- et al.
Three-dimensional cerebral vasculature of the CBA mouse brain: a magnetic resonance imaging and micro computed tomography study
NeuroImage
(2007) - et al.
High resolution three-dimensional brain atlas using an average magnetic resonance image of 40 adult C57Bl/6J mice
NeuroImage
(2008) - et al.
Extracting branching tubular object geometry via cores
Med. Image Anal.
(2004) - et al.
A new software correction approach to volume averaging artifacts in CT
Comput. Radiol.
(1986) - et al.
Hierarchical microimaging for multiscale analysis of large vascular networks
NeuroImage
(2006) - et al.
Three distinct auditory areas of cortex (AI, AII, and AAF) defined by optical imaging of intrinsic signals
NeuroImage
(2000) - et al.
Multisensory plasticity in congenitally deaf mice: how are cortical areas functionally specified?
Neuroscience
(2006) - et al.
Vasa vasorum neovascularization and lesion distribution among different vascular beds in ApoE(−/−)/LDL−/− double knockout mice
Atherosclerosis
(2007) - et al.
Cortical thickness measured from MRI in the YAC128 mouse model of Huntington's disease
NeuroImage
(2008) - et al.
Morphometry of the human cerebral cortex microcirculation: general characteristics and space-related profiles
NeuroImage
(2008)
Successive depth variations in microvascular distribution of rat somatosensory cortex
Brain Res.
3D visualisation and quantification by microcomputed tomography of late gestational changes in the arterial and venous feto-placental vasculature of the mouse
Placenta
Sexual dimorphism revealed in the structure of the mouse brain using three-dimensional magnetic resonance imaging
NeuroImage
Variation in the cortical area map of C57BL/6J and DBA/2J inbred mice predicts strain identity
BMC Neurosci.
Optimization of intraperitoneal injection anesthesia in mice: drugs, dosages, adverse effects, and anesthesia depth
Comp. Med.
Brain microvascular changes in Alzheimer's disease and other dementias
Ann. N.Y. Acad. Sci.
Cited by (98)
Brain microvascular damage linked to a moderate level of strain induced by controlled cortical impact
2021, Journal of BiomechanicsA TfR-Binding Cystine-Dense Peptide Promotes Blood–Brain Barrier Penetration of Bioactive Molecules
2020, Journal of Molecular BiologyUltrasound Detection of Abnormal Cerebrovascular Morphology in a Mouse Model of Sickle Cell Disease Based on Wave Reflection
2019, Ultrasound in Medicine and BiologyCitation Excerpt :The pulsatility index (PI) was calculated as the difference between peak systolic and end diastolic velocities, divided by the mean velocity over the cardiac cycle. All of the micro-CT images were registered to a common space using vascular landmarks and a 3-D magnetic resonance imaging anatomic brain atlas (Dorr et al. 2008), as previously described (Chugh et al. 2009). Using Display (MINC toolkit, McConnell Brain Imaging Centre, Montreal, QC, Canada), each image was manually segmented to exclude extra-cerebral vessels.
Advances in imaging feto-placental vasculature: New tools to elucidate the early life origins of health and disease
2021, Journal of Developmental Origins of Health and Disease