Noninvasive and high spatiotemporal resolution imaging of cerebral hemodynamic and neuronal effects in response to various types of stimulations (e.g., electrical stimulations, drug challenges) remains a major challenge in neuroimaging. While LSI permits 2D imaging of CBF at high spatiotemporal resolutions (e.g., 30μm, 10Hz), it is based on en face imaging and only measures the relative flow indices rather than the absolute flow rates. Doppler OCT is an emerging optical technique that enables quantitative 3D imaging of the vascular CBF network (absolute flow rate detection) at high spatial resolution (~10μm) over a large FOV in the cerebral cortex of the rodent’s brain. Recent advances in DFR-OCT have dramatically improved the sensitivity of Doppler OCT for detection of cerebral capillary flow (e.g., ~10μm, 0.16mm/s) and the frame rate needed to render 3D imaging of the vascular CBF network within 8s/volume (e.g., ); however, post image processing is needed because of the intensive computation required to reconstruct quantitative 3D DFR-OCT flow images. By co-registering with DFR-OCT, it is found that LSI can be calibrated (e.g., ) to allow for high-resolution absolute quantitative imaging of transient CBF changes in real time (e.g., 10–29Hz). Moreover, DW-LSI can measure changes in both [HbO2] and [HbR], thus enabling determination of the change in total hemoglobin concentration (i.e., Δ [HbT] or Δ CBV) in both CBFs and tissue perfusion (i.e., irresolvable capillary flows). Therefore, a multimodality neuroimaging platform that combines DFR-OCT, DW-LSI and fluorescence can allow for simultaneous characterization of the local changes in cerebrovascular hemodynamics (CBF, CBV), hemoglobin oxygenation (HbO2) and intracellular calcium ([Ca2+]i fluorescence) as shown here for monitoring the effects of cocaine. Such a multimodality imaging technique (OFI) provides several uniquely important merits, including: 1) large FOV (~3 × 5mm2), 2) high spatiotemporal resolutions (~30μm, ~10Hz), 3) quantitative 3D imaging of the CBF network by co-registering with DFR-OCT, 4) label-free imaging of hemodynamic changes, 5) separation of vascular compartments between arterial and venous vessels and monitoring of cortical brain metabolic changes, 6) simultaneous imaging and thus separation of cellular (neuronal) from vascular responses, and 7) the ability to separately measure CBF in the layers of the cerebral cortex in the rodent brain.
The animal study presented here validates the technological feasibility of this multi-modal approach for simultaneous imaging. Experimental results presented in – demonstrate the utility of this new technique to enable in vivo imaging of rat cortical brain functional changes in response to cocaine administration at high spatiotemporal resolutions over a large of field of view. The critical significance of such imaging modalities on neuroimaging and the drug-induced pharmacologic effects on the brain function can be summarized as follows:
- Full-Field and high spatial resolution: DW-LSI enables imaging of the vascular CBF network at high spatial resolution (~30μm) and over a large FOV (4 × 5mm2). By co-registering with DFR-OCT, quantitative 3D imaging of the CBF network is permitted over a cubic cortical volume of 2.5 × 2 × 2.5mm3 at ~10μm resolution. Such high spatial resolution and large FOV will bridge the gap between conventional mesoscopic (e.g., fMRI, PET) and microscopic (e.g., confocal, 2-photon) imaging modalities to study cerebral neurovascular coupling effects which require both high resolution and large field of view.
- High temporal resolution: The frame rates of our current DW-LSI and 2D DFR-OCT are 10fps and 47fps and can be easily upgraded to 30fps and 94fps (i.e., 0.25Hz for 3D DFR-OCT). Such high temporal-resolution 2D and 3D CBF imaging enables to capture transient neurovascular events (as shown in this study for the case of cocaine) to provide new insights to advance our understanding of the coupling between neuronal activity, metabolism and hemodynamic changes and the perturbation by pharmacological agents and animal models of brain diseases. Additionally, it will likely allow us to monitor the treatment or therapeutic effects aimed at vascular as well as neuronal repair.
- Multimodality simultaneous imaging: The optical/fluorescence techniques that combine DW-LSI, 3D DFR-OCT and [Ca2+]i fluorescence enable concurrent imaging of changes in hemodynamics (CBF, CBV), hemoglobin oxygenation (HbO2) and [Ca2+]i from cortical brain in vivo. The value for simultaneous imaging of neurovascular and cellular functions is illustrated by our results with cocaine that clearly differentiated cocaine-induced cellular (neuronal) effects from its neurovascular effects.
The findings we report here with cocaine corroborate and advance our prior studies that measured the effects of cocaine in local cerebral hemodynamics (CBV), oxygenation and [Ca2+
fluorescence). In our prior optical spectroscopy study we showed that acute cocaine (e.g., 1mg/kg, i.v.) induced a local increase in CBV and hemoglobin oxygenation in rats (α-chloralose anesthesia)(Du et al., 2006
). The current study advances our findings of cocaine’s effects on the brain in the following 2 important aspects. First, we advance from point measurement (acquiring averaged signal across a brain area defined by fiberoptic probe, e.g.,
=5mm of our prior spectroscope) to high-resolution imaging. With DW-LSI, we achieve high spatiotemporal resolutions (e.g., ~30μm and ~10Hz) which allow us to resolve individual vascular compartments and distinguish the vascular effects (i.e., changes in CBF, [HbR], [HbO2
] and CBV) from the cellular effects (i.e., changes in [Ca2+
fluorescence) induced by cocaine in the cortical brain; Secondly, we integrate three imaging techniques (DW-LSI, DFR-OCT, and [Ca2+
fluorescence) into a multimodal imaging platform (OFI) to enable tri-modal simultaneous imaging. By co-registering with DFR-OCT, the 3D CBF network can be quantitatively ‘visualized’ across various depths of a rodent cortical brain (e.g., up to z=1mm in a rat brain) and image their changes in response to cocaine administration. The image results using DW-LSI and DFR-OCT show more detailed cocaine-induced effects. For instance, we observed the late phase (phase 2) of cocaine’s neurovascular effect, e.g., increases in CBF (–), CBV or [HbT] and [HbO2
] along with a decrease of [HbR] () after 4–5min of cocaine injection with single-vessel resolution. During this time period, DW-LSI showed an increase in [Ca2+
fluorescence with concurrent CBF elevation induced by cocaine, in agreement with prior studies(Du et al., 2006
; Hu, 2007
; Lu et al., 2007
; Nasif et al., 2005
). Importantly, the high temporal resolution and simultaneous imaging capability of our OFI allowed us to visualize the early phase (phase 1) of cocaine’s neurovascular effect, e.g., an immediate early increase in [Ca2+
along with an immediate transient decrease in CBF during the first 3.5 ± 0.9min (n=4) following cocaine administration (e.g., ). Indeed, the early transient CBF ’dip’ observed in this study is consistent with the report of sharp negative BOLD signal measured with fMRI at up to 120s after cocaine administration in rat cortical brain (urethane anesthesia)(Luo et al., 2003
), which was interpreted to reflect neuronal-based vascular constriction induced by cocaine. Our findings of an immediate increase in [Ca2+
(indicator of neuronal activation) with a concomitant decrease in CBF and in [HbO2
] and increase in [HbR] and CBV in arteriole (, upper left panel) suggest that the transient negative BOLD signal reported with fMRI shortly after cocaine reflects a decrease in hemoglobin oxygenation secondary to the temporally lagging in CBF response to the increases in neuronal activation elicited by cocaine. Cocaine’s immediate increases in neuronal activity and abrupt decrease (dip) in CBF and [HbO2
] could underlie the cerebrovascular complications associated with cocaine abuse, e.g., ischemic stroke(Johnson et al., 2001
). The findings of cocaine-induced Ca2+
increases could also underlie the reported enhanced hemodynamic and field potential responses to sensory stimulation after acute cocaine administration(Devonshire et al., 2004
). It is noteworthy that a sampling rate of 10Hz in this study was not fast enough to capture the calcium transients of individual neuron firings. However, unlike somatic electrical stimulation, cocaine directly stimulates neuronal activity and neuron firings might not necessarily synchronize, which would make it difficult to distinguish even at a higher frame rate (e.g., 30fps). The cocaine-induced mean [Ca2+
increase measured here () could reflect the amplitude increase in neuronal Ca transients or the increase in neuronal firing rate or a combination of both.
Recent technological advances in OCT angiography have dramatically improved the sensitivity for Doppler flow imaging so that subsurface minute blood flows, including capillary flows that can be uncovered (Mariampillai et al., 2010
; Srinivasan et al., 2010
; Wang et al., 2007
). For instance, shows a maximum-intensity projection of a full-field 3D OCT angiography of a mouse cortex which resolves far more detailed cerebral microvasculature than white-light surface imaging. Noteworthily, despite more microvasculature seen in than in DFR-OCT (e.g., –), OCT angiography is unable to provide quantitative flow changes crucial to brain functional studies such as cocaine effects presented in this work. Therefore, our future work will combine quantitative DFR-OCT with OCT angiography to lift the limitation. Additionally, two wavelengths (785nm, 830nm) close to the isosbestic point of hemoglobin absorption (805nm) were chosen for DW-LSI, which might benefit to increase the flow imaging depth (due to reduced tissue scattering with wavelength) and alleviate complications in Δ[HbO2
] and Δ [HbR] calculation in Eq.(2)
induced by pathlength difference between these two wavelengths. However, the low hemoglobin absorption may lead to reduced sensitivity in detecting HbO2
changes. Alternative approaches include using 830nm for LSI and decoupled reflectance images at other wavelengths(Kawauchi et al., 2009
; Okui and Okada, 2005
; Strangman et al., 2003
; Uludag et al., 2004
) with a stronger HbO2
absorbance (e.g., 690nm) for Δ [HbO2
] detection, which in return might encounter potential problems such as reduced depth for flow imaging and complications in Δ [HbO2
] computation and image registration with LSI. Nevertheless, it will be interesting to compare the advantages and limitations between these approaches.
Fig. 6 Enhanced cerebral microvasculature with 3D OCT angiography. A) Surface image of a mouse cranial window. B) Maximum intensity projection of a full-field 3D OCT angiography within the cranial window. Although not quantitative, OCT angiography was able to (more ...)
In summary, we present a multimodality optical imaging technique which combines DW-LSI, DFR-OCT and Rhod2–labeled [Ca2+]i fluorescence. Results of in vivo animal studies demonstrate the potential of such imaging platform for simultaneous imaging and absolute quantification of changes in neuronal, metabolic and hemodynamic parameters of the cortical brain. More specifically, it allows us: 1) to analyze the temporal effects of cocaine on neurovascular network and oxygenation; 2) to delineate the dynamic processes that occur between the vascular CBF network and the surrounding neuronal tissue and to separately evaluate cellular from vascular effects by comparing the transient changes between [Ca2+]i fluorescence from [HbO2] and CBF; and 3) to do in quantitative CBF measures at different depths in the cortex. Using this multimodality imaging platform we show a transient response to cocaine that revealed an immediate and transient decrease (phase 1) in local oxygen content () and CBF (t<4min) followed by a longer lasting overshoot (up to 40min, phase 2) whereas cocaine induced an immediate Ca2+ increase (peaked at 4.1 ± 0.4min) that remained elevated over 20min of the measurements (). This identifies a 2.9 ± 0.5min lagging time between the vascular and the neuronal responses to cocaine. This method complements other existing neuroimaging approaches for use in neuroscience research including that of the investigation of the coupling between neuronal activation and hemodynamic and metabolic responses of cerebral tissue.