Simultaneous two-photon imaging of oxygen and blood flow in deep cerebral vessels. Nat Med

Institut National de Santé et de Recherche Médicale (INSERM), U603, Paris, France.
Nature medicine (Impact Factor: 27.36). 06/2011; 17(7):893-8. DOI: 10.1038/nm.2394
Source: PubMed


Uncovering principles that regulate energy metabolism in the brain requires mapping of partial pressure of oxygen (PO(2)) and blood flow with high spatial and temporal resolution. Using two-photon phosphorescence lifetime microscopy (2PLM) and the oxygen probe PtP-C343, we show that PO(2) can be accurately measured in the brain at depths up to 300 μm with micron-scale resolution. In addition, 2PLM allowed simultaneous measurements of blood flow and of PO(2) in capillaries with less than one-second temporal resolution. Using this approach, we detected erythrocyte-associated transients (EATs) in oxygen in the rat olfactory bulb and showed the existence of diffusion-based arterio-venous shunts. Sensory stimulation evoked functional hyperemia, accompanied by an increase in PO(2) in capillaries and by a biphasic PO(2) response in the neuropil, consisting of an 'initial dip' and a rebound. 2PLM of PO(2) opens new avenues for studies of brain metabolism and blood flow regulation.

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Available from: Mathieu Ducros, Oct 10, 2015
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    • "Pellerin and Magistretti, 1994; Bélanger et al., 2011), that research to understand the fine detail of neuron-astrocyte communication is so important for understanding functional brain imaging signals. Studies in which the activity of neurons and astrocytes can be differentiated and/or specifically manipulated , for example using optogenetic approaches combined with 2PLSM will be significant in this regard (Li et al., 2013a) and combination of these approaches with metabolic readouts, for instance using molecular oxygen sensors (Lecoq et al., 2011) or more macroscopic techniques (e.g. see Devor et al., 2012) will advance understanding considerably. "
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    ABSTRACT: Many of the major advances in our understanding of how functional brain imaging signals relate to neuronal activity over the previous two decades have arisen from physiological research studies involving experimental animal models. This approach has been successful partly because it provides opportunities to measure both the hemodynamic changes that underpin many human functional brain imaging techniques and the neuronal activity about which we wish to make inferences. Although research into the coupling of neuronal and hemodynamic responses using animal models has provided a general validation of the correspondence of neuroimaging signals to specific types of neuronal activity, it is also highlighting the key complexities and uncertainties in estimating neural signals from hemodynamic markers. This review will detail how research in animal models is contributing to our rapidly evolving understanding of what human neuroimaging techniques tell us about neuronal activity. It will highlight emerging issues in the interpretation of neuroimaging data that arise from in vivo research studies, for example spatial and temporal constraints to neuroimaging signal interpretation, or the effects of disease and modulatory neurotransmitters upon neurovascular coupling. We will also give critical consideration to the limitations and possible complexities of translating data acquired in the typical animals models used in this area to the arena of human fMRI. These include the commonplace use of anesthesia in animal research studies and the fact that many neuropsychological questions that are being actively explored in humans have limited homologs within current animal models for neuroimaging research. Finally we will highlighting approaches, both in experimental animals models (e.g. imaging in conscious, behaving animals) and human studies (e.g. combined fMRI-EEG), that mitigate against these challenges.
    Frontiers in Neuroscience 08/2014; 8(8):211. DOI:10.3389/fnins.2014.00211 · 3.66 Impact Factor
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    • "To the best of our knowledge, the nature and frequency of artificial surgical damage have not been reported in detail in mice or in rats following the removal of dura mater [21,23,25,26,30-32,38-41]. Thus, we cannot discuss our findings in relation to the data of other research groups. "
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    ABSTRACT: There is growing evidence that endothelial failure and subsequent blood brain barrier (BBB) breakdown initiate cerebral small vessel disease (CSVD) pathology. In spontaneously hypertensive stroke-prone rats (SHRSP) endothelial damage is indicated by intraluminal accumulations of erythrocytes (erythrocyte thrombi) that are not observed with current magnetic resonance imaging techniques. Two-photon microscopy (2 PM) offers the potential for real-time direct detection of the small vasculature. Thus, within this pilot study we investigated the sensitivity of 2 PM to detect erythrocyte thrombi expressing initiating CSVD phenomena in vivo. Eight SHRSP and 13 Wistar controls were used for in vivo imaging and subsequent histology with haematoxylin-eosin (HE). For 2 PM, cerebral blood vessels were labeled by fluorescent Dextran (70 kDa) applied intraorbitally. The correlation between vascular erythrocyte thrombi observed by 2 PM and HE-staining was assessed. Artificial surgical damage and parenchymal Dextran distribution were analyzed postmortem. Dextran was distributed within the small vessel walls and co-localized with IgG.Artificial surgical damage was comparable between SHRSP and Wistar controls and mainly affected the small vasculature. In fewer than 20% of animals there was correlation between erythrocyte thrombi as observed with 2 PM and histologically with HE. Contrary to our initial expectations, there was little agreement between intravital 2 PM imaging and histology for the detection of erythrocyte thrombi. Two-photon microscopy is a valuable technique that complements but does not replace the value of conventional histology.
    Experimental and Translational Stroke Medicine 01/2014; 6(1):1. DOI:10.1186/2040-7378-6-1
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    • "These properties combined with its phosphorescence efficiency make PtP-C343 an effectively brighter and stable molecular probe for two-photon microscopy, resulting in greater confidence in measurements and improved three-dimensional sectioning. The new oxygen sensor has shown convincing calibration in vitro and in vivo through blood gas analysis along with application in a series of baseline [9,10] and functional activation [11,12] pO2 measurements in intravascular and interstitial tissue environments. Here, we demonstrate a two-photon lifetime microscopy technique that utilizes this new oxygen sensor for examining the vascular networking impact on intravascular oxygen tension. "
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    ABSTRACT: Occlusions in single cortical microvessels lead to a reduction in oxygen supply, but this decrement has not been able to be quantified in three dimensions at the level of individual vessels using a single instrument. We demonstrate a combined optical system using two-photon phosphorescence lifetime and fluorescence microscopy (2PLM) to characterize the partial pressure of oxygen (pO2) in single descending cortical arterioles in the mouse brain before and after generating a targeted photothrombotic occlusion. Integrated real-time Laser Speckle Contrast Imaging (LSCI) provides wide-field perfusion maps that are used to monitor and guide the occlusion process while 2PLM maps changes in intravascular oxygen tension. We present the technique’s utility in highlighting the effects of vascular networking on the residual intravascular oxygen tensions measured after occlusion in three dimensions.
    Biomedical Optics Express 07/2013; 4(7):1061-1073. DOI:10.1364/BOE.4.001061 · 3.65 Impact Factor
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