Emiri T. Mandeville

OCT methods for capillary velocimetry

To date, two main categories of OCT techniques have been described for imaging hemodynamics: Doppler OCT and OCT angiography. Doppler OCT can measure axial velocity profiles and flow in arteries and veins, while OCT angiography can determine vascular morphology, tone, and presence or absence of red blood cell (RBC) perfusion. However, neither method can quantify RBC velocity in capillaries, where RBC flow is typically transverse to the probe beam and single-file. Here, we describe new methods that potentially address these limitations. Firstly, we describe a complex-valued OCT signal in terms of a static scattering component, dynamic scattering component, and noise. Secondly, we propose that the time scale of random fluctuations in the dynamic scattering component are related to red blood cell velocity. Analysis was performed along the slow axis of repeated B-scans to parallelize measurements. We correlate our purported velocity measurements against two-photon microscopy measurements of RBC velocity, and investigate changes during hypercapnia. Finally, we image the ischemic stroke penumbra during distal middle cerebral artery occlusion (dMCAO), where OCT velocimetry methods provide additional insight that is not afforded by either Doppler OCT or OCT angiography.

Large arteriolar component of oxygen delivery implies a safe margin of oxygen supply to cerebral tissue

What is the organization of cerebral microvascular oxygenation and morphology that allows adequate tissue oxygenation at different activity levels? We address this question in the mouse cerebral cortex using microscopic imaging of intravascular O2 partial pressure and blood flow combined with numerical modeling. Here we show that parenchymal arterioles are responsible for 50% of the extracted O2 at baseline activity and the majority of the remaining O2 exchange takes place within the first few capillary branches. Most capillaries release little O2 at baseline acting as an O2 reserve that is recruited during increased neuronal activity or decreased blood flow. Our results challenge the common perception that capillaries are the major site of O2 delivery to cerebral tissue. The understanding of oxygenation distribution along arterio-capillary paths may have profound implications for the interpretation of BOLD fMRI signal and for evaluating microvascular O2 delivery capacity to support cerebral tissue in disease.

Quantifying the Microvascular Origin of BOLD-fMRI from First Principles with Two-Photon Microscopy and an Oxygen-Sensitive Nanoprobe

The blood oxygenation level-dependent (BOLD) contrast is widely used in functional magnetic resonance imaging (fMRI) studies aimed at investigating neuronal activity. However, the BOLD signal reflects changes in blood volume and oxygenation rather than neuronal activity per se. Therefore, understanding the transformation of microscopic vascular behavior into macroscopic BOLD signals is at the foundation of physiologically informed noninvasive neuroimaging. Here, we use oxygen-sensitive two-photon microscopy to measure the BOLD-relevant microvascular physiology occurring within a typical rodent fMRI voxel and predict the BOLD signal from first principles using those measurements. The predictive power of the approach is illustrated by quantifying variations in the BOLD signal induced by the morphological folding of the human cortex. This framework is then used to quantify the contribution of individual vascular compartments and other factors to the BOLD signal for different magnet strengths and pulse sequences.

Dendritic upconverting nanoparticles enable in vivo multiphoton microscopy with low-power continuous wave sources

We report a group of optical imaging probes, comprising upconverting lanthanide nanoparticles (UCNPs) and polyanionic dendrimers. Dendrimers with rigid cores and multiple carboxylate groups at the periphery are able to tightly bind to surfaces of UCNPs pretreated with NOBF4, yielding stable, water-soluble, biocompatible nanomaterials. Unlike conventional linear polymers, dendrimers adhere to UCNPs by donating only a fraction of their peripheral groups to the UCNP–surface interactions. The remaining termini make up an interface between the nanoparticle and the aqueous phase, enhancing solubility and offering multiple possibilities for subsequent modification. Using optical probes as dendrimer cores makes it possible to couple the UCNPs signal to analyte-sensitive detection via UCNP-to-chromophore excitation energy transfer (EET). As an example, we demonstrate that UCNPs modified with porphyrin–dendrimers can operate as upconverting ratiometric pH nanosensors. Dendritic UCNPs possess excellent photostability, solubility, and biocompatibility, which make them directly suitable for in vivo imaging. Polyglutamic dendritic UCNPs injected in the blood of a mouse allowed mapping of the cortical vasculature down to 400 μm under the tissue surface, thus demonstrating feasibility of in vivo high-resolution two-photon microscopy with continuous wave (CW) excitation sources. Dendrimerization as a method of solubilization of UCNPs opens up numerous possibilities for use of these unique agents in biological imaging and sensing.

Oxidative stress interferes with white matter renewal after prolonged cerebral hypoperfusion in mice.

Background and Purpose— White matter injury caused by cerebral hypoperfusion may contribute to the pathophysiology of vascular dementia and stroke, but the underlying mechanisms remain to be fully defined. Here, we test the hypothesis that oxidative stress interferes with endogenous white matter repair by disrupting renewal processes mediated by oligodendrocyte precursor cells (OPCs). Methods— In vitro, primary rat OPCs were exposed to sublethal CoCl2 for 7 days to induce prolonged chemical hypoxic stress. Then, OPC proliferation/differentiation was assessed. In vivo, prolonged cerebral hypoperfusion was induced by bilateral common carotid artery stenosis in mice. Then, reactive oxygen species production, myelin density, oligodendrocyte versus OPC counts, and cognitive function were evaluated. To block oxidative stress, OPCs and mice were treated with the radical scavenger edaravone. Results— Prolonged chemical hypoxic stress suppressed OPC differentiation in vitro. Radical scavenging with edaravone ameliorated these effects. After 28 days of cerebral hypoperfusion in vivo, reactive oxygen species levels were increased in damaged white matter, along with the suppression of OPC-to-oligodendrocyte differentiation and loss of myelin staining. Concomitantly, mice showed functional deficits in working memory. Radical scavenging with edaravone rescued OPC differentiation, ameliorated myelin loss, and restored working memory function. Conclusions— Our proof-of-concept study demonstrates that after prolonged cerebral hypoperfusion, oxidative stress interferes with white matter repair by disrupting OPC renewal mechanisms. Radical scavengers may provide a potential therapeutic approach for white matter injury in vascular dementia and stroke.

Inhibition of 12/15-lipoxygenase as therapeutic strategy to treat stroke.

Targeting newly identified damage pathways in the ischemic brain can help to circumvent the currently severe limitations of acute stroke therapy. Here we show that the activity of 12/15‐lipoxygenase was increased in the ischemic mouse brain, and 12/15‐lipoxygenase colocalized with a marker for oxidized lipids, MDA2. This colocalization was also detected in the brain of 2 human stroke patients, where it also coincided with increased apoptosis‐inducing factor. A novel inhibitor of 12/15‐lipoxygenase, LOXBlock‐1, protected neuronal HT22 cells against oxidative stress. In a mouse model of transient focal ischemia, the inhibitor reduced infarct sizes both 24 hours and 14 days poststroke, with improved behavioral parameters. Even when treatment was delayed until at least 4 hours after onset of ischemia, LOXBlock‐1 was protective. Furthermore, it reduced tissue plasminogen activator‐associated hemorrhage in a clot model of ischemia/reperfusion. This study establishes inhibition of 12/15‐lipoxygenase as a viable strategy for first‐line stroke treatment. Ann Neurol 2013

Neuronal Production of Lipocalin-2 as a Help-Me Signal for Glial Activation

Background and Purpose— We explored the hypothesis that injured neurons release lipocalin-2 as a help me signal. Methods— In vivo lipocalin-2 responses were assessed in rat focal cerebral ischemia and human stroke brain samples using a combination of ELISA and immunostaining. In vitro, microglia and astrocytes were exposed to lipocalin-2, and various markers and assays of glial activation were quantified. Functional relevance of neuron-to-glia lipocalin-2 signaling was examined by transferring conditioned media from lipocalin-2–activated microglia and astrocytes onto neurons to see whether activated glia could protect neurons against oxygen–glucose deprivation and promote neuroplasticity. Results— In human stroke samples and rat cerebral ischemia, neuronal expression of lipocalin-2 was significantly increased. In primary cell cultures, exposing microglia and astrocytes to lipocalin-2 resulted in glial activation. In microglia, lipocalin-2 converted resting ramified shapes into a long-rod morphology with reduced branching, increased interleukin-10 release, and enhanced phagocytosis. In astrocytes, lipocalin-2 upregulated glial fibrillary acid protein, brain-derived neurotropic factor, and thrombospondin-1. Conditioned media from lipocalin-2–treated astrocytes upregulated synaptotagmin, and conditioned media from lipocalin-2–treated microglia upregulated synaptophysin and post-synaptic density 95 (PSD95) and protected neurons against oxygen–glucose deprivation. Conclusions— These findings provide proof of concept that lipocalin-2 is released by injured neurons as a help me distress signal that activates microglia and astrocytes into potentially prorecovery phenotypes.

Multiparametric, Longitudinal Optical Coherence Tomography Imaging Reveals Acute Injury and Chronic Recovery in Experimental Ischemic Stroke

Progress in experimental stroke and translational medicine could be accelerated by high-resolution in vivo imaging of disease progression in the mouse cortex. Here, we introduce optical microscopic methods that monitor brain injury progression using intrinsic optical scattering properties of cortical tissue. A multi-parametric Optical Coherence Tomography (OCT) platform for longitudinal imaging of ischemic stroke in mice, through thinned-skull, reinforced cranial window surgical preparations, is described. In the acute stages, the spatiotemporal interplay between hemodynamics and cell viability, a key determinant of pathogenesis, was imaged. In acute stroke, microscopic biomarkers for eventual infarction, including capillary non-perfusion, cerebral blood flow deficiency, altered cellular scattering, and impaired autoregulation of cerebral blood flow, were quantified and correlated with histology. Additionally, longitudinal microscopy revealed remodeling and flow recovery after one week of chronic stroke. Intrinsic scattering properties serve as reporters of acute cellular and vascular injury and recovery in experimental stroke. Multi-parametric OCT represents a robust in vivo imaging platform to comprehensively investigate these properties.

Deficient eNOS Phosphorylation Is a Mechanism for Diabetic Vascular Dysfunction Contributing to Increased Stroke Size

Background and Purpose— Phosphorylation of eNOS, an important post-translational modulator of its enzymatic activity, is reduced in diabetes mellitus. We hypothesized that modulation of eNOS phosphorylation could overcome diabetic vascular dysfunction and improves the outcome to stroke. Methods— We used the db/db mouse model of type 2 diabetes mellitus. We mated db/db mice with eNOS knock-in mice that carry single amino acid mutations at the S1176 phosphorylation site; the phosphomimetic SD mutation (serine replaced by aspartate) shows increased eNOS enzymatic activity, whereas the unphosphorylatable SA mutation (serine replaced by alanine) shows decreased eNOS activity. We characterized the vascular anatomy, baseline physiological parameters, and vascular reactivity. We used the middle cerebral artery occlusion model of stroke and measured infarct volume and neurological deficits. Results— db/db mice showed diminished eNOS phosphorylation at S1176. eNOS SD and SA mutations do not change the vascular anatomy at the Circle of Willis, brain capillary density, heart rate, or arterial blood gases of db/db mice. The eNOS SD mutation, but not the SA mutation, lowers blood pressure and improves vascular reactivity to acetylcholine in db/db mice. The eNOS SD mutation reduces stroke size and neurological deficit after middle cerebral artery occlusion. Conclusions— Diminished eNOS phosphorylation is a mechanism of vascular dysfunction in db/db mice. We show here that modulation of the eNOS S1176 phosphorylation site in db/db mice is associated with improved vascular reactivity and improved outcome to stroke after middle cerebral artery occlusion.

Optical coherence tractography using intrinsic contrast

Organs such as the heart and brain possess intricate fiber structures that are best characterized with three-dimensional imaging. For instance, diffusion-based, magnetic resonance tractography (MRT) enables studies of connectivity and remodeling during development and disease macroscopically on the millimeter scale. Here we present complementary, high-resolution microscopic optical coherence imaging and analysis methods that, when used in conjunction with clearing techniques, can characterize fiber architecture in intact organs at tissue depths exceeding 1 mm. We anticipate that these techniques can be used to study fiber architecture in situ at microscopic scales not currently accessible to diffusion magentic resonance (MR), and thus, to validate and complement macroscopic structural imaging techniques. Moreover, as these techniques use intrinsic signals and do not require tissue slicing and staining, they can be used for high-throughput, nondestructive evaluation of fiber architecture across large tissue volumes.