Depth-resolved optical imaging and microscopy of vascular compartment dynamics during somatosensory stimulation
Introduction
The cortical hemodynamic response to stimulus provides a detectable signal which can report the presence and location of neuronal activation (Fox and Raichle, 1986, Woolsey et al., 1996). Functional Magnetic Resonance imaging (fMRI) provides an unsurpassed tool with which to image the hemodynamic response non-invasively and in a clinical setting (Belliveau et al., 1991). However, interpretation of fMRI data is confounded by relatively poor specificity to neuronal events: The MRI-based blood oxygen level dependent (BOLD) signal is primarily sensitive to deoxy-hemoglobin concentration changes, and originates in both the capillary beds of the cortex as well as in more distant draining veins (Mandeville et al., 1999, Turner, 2002).
Consensus has also yet to be reached about the neurovascular mechanisms by which the cortical hemodynamic response arises, both in terms of how it is triggered and controlled, and how it proceeds as the stimulus response evolves (Attwell and Iadecola, 2002, Iadecola, 2004). To date, the hemodynamic response has largely just been considered as the sum total of all the activity observed that temporally correlates with a presented stimulus (Arthurs and Boniface, 2002, Iadecola, 2002). In this paper we utilize the well characterized rat forepaw stimulus model and apply novel optical imaging technologies to investigate the hemodynamic response in detail. Using high resolution optical imaging and microscopy, we are able to resolve the behavior of the individual vascular compartments involved in the ensemble hemodynamic response. By investigating the role of each compartment, we are able to more fully understand both the composition, and basic mechanisms of the observable hemodynamic signal.
Optical imaging of the exposed cortex not only provides high spatial resolution, but also high frame rates and simultaneous sensitivity to oxy-, deoxy- and total hemoglobin changes (HbO2, HbR and HbT) (Devor et al., 2003, Hewson-Stoate et al., 2005, Malonek and Grinvald, 1996, Sheth et al., 2005). However, camera-based optical imaging only provides 2-dimensional (2D) images of the cortical surface, making it difficult to quantify and distinguish between signals from superficial vasculature and deeper capillary beds. In this paper, we utilize a recently developed optical imaging technology called Laminar Optical Tomography (LOT) (Hillman et al., 2004), which is capable of high-resolution depth-resolved imaging of hemodynamic activity in the cortex to depths of around 2 mm. We complement LOT with rapid, full-field two photon microscopy, both to validate our LOT findings, and to further explore the vascular mechanisms of the hemodynamic response in-vivo.
Using LOT, we demonstrate that it is possible to distinguish subtle yet distinctive functional temporal signatures associated with the hemodynamic response in the different vascular compartments. We show that these signatures can be used to spatially resolve the locations of the different compartments in the cortex in 3-dimensions (3D). We validate our findings with two-photon microscopy of vascular casts from the same rats. We further demonstrate that these differences in the vascular compartment responses are robust, and have potential as a mechanism for spatiotemporal separation of the vascular compartments in other functional imaging modalities including fMRI.
The extracted temporal signatures of the vascular compartments also provide new insights into the vascular mechanisms taking place. We utilize in-vivo two-photon microscopy of vessels in the brain during somatosensory stimulus to further validate and explore the physical vascular mechanisms that underlie the distinct temporal characteristics of the functional response in each compartment.
We present evidence that arteriolar dilation dynamics correlate strongly with the blood flow response in the veins. We further show that, for 4 s stimuli, the most significant changes in the venous compartment are speed of flow and oxygenation changes, and not venous dilation or ballooning. We utilize our results to revisit standing theories and assumptions about neurovascular coupling and how the hemodynamic response is controlled, including capillary hyperemia and directional propagation of vasodilation. We discuss the implications of our findings to hemodynamic modeling and to other functional imaging modalities.
Section snippets
Methods
In-vivo imaging experiments were performed using rats undergoing 4-s-long electrical forepaw stimulation (Brecht et al., 2003, Jones et al., 2001, Mandeville et al., 1999, Pain et al., 2002, Sheth et al., 2005, Shibuki et al., 2003, Silva and Koretsky, 2002). Detailed procedures are described below.
Depth-resolved imaging of the hemodynamic response
Fig. 2 shows depth-resolved cortical functional activation maps from one rat, acquired using LOT during electrical forepaw stimulation; Fig. 2A shows a CCD camera image of the field of view, acquired with 570 nm illumination prior to LOT imaging (the LOT field of view is identified by the dotted white square). Fig. 2B shows horizontal LOT slices of the depth-resolved HbR, HbO2 and HbT functional changes 0.6 s after cessation of the stimulus (block-average of 140 stimuli). The most superficial
Discussion
There is widespread disagreement in the literature regarding the physical properties and behavior of the vascular compartments during functional stimulation. Partly, this has arisen from a reliance on historical ex-vivo and in-vitro data, and the combination of conclusions drawn from very different experiments, using different measurement modalities (fMRI, optical, laser Doppler) and stimuli of different types, strengths and durations. Our extraction of the individual timecourses of HbR, HbO2
Acknowledgments
This work was funded by R21NS053684, R01EB000790, K25NS41291, R01EB000768, R01NS05118 and The Whitaker Foundation and the Wallace H Coulter Foundation. The authors wish to thank Svetlana Ruvinskaya, Alan Koretsky, Alfonso Silva, Britton Chance, Edith Hamel, Bruno Cauli, Noam Harel, Joseph Mandeville, Jens Steinbrink, David Attwell, Nozomi Nishimura and David Kleinfeld for useful advice and discussions.
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