A deep learning approach to 3D segmentation of brain vasculature

The segmentation of blood-vessels is an important preprocessing step for the quantitative analysis of brain vasculature. We approach the segmentation task for two-photon brain angiograms using a fully convolutional 3D deep neural network.Published versio

Awake chronic mouse model of targeted pial vessel occlusion via photothrombosis

Animal models of stroke are used extensively to study the mechanisms involved in the acute and chronic phases of recovery following stroke. A translatable animal model that closely mimics the mechanisms of a human stroke is essential in understanding recovery processes as well as developing therapies that improve functional outcomes. We describe a photothrombosis stroke model that is capable of targeting a single distal pial branch of the middle cerebral artery with minimal damage to the surrounding parenchyma in awake head-fixed mice. Mice are implanted with chronic cranial windows above one hemisphere of the brain that allow optical access to study recovery mechanisms for over a month following occlusion. Additionally, we study the effect of laser spot size used for occlusion and demonstrate that a spot size with small axial and lateral resolution has the advantage of minimizing unwanted photodamage while still monitoring macroscopic changes to cerebral blood flow during photothrombosis. We show that temporally guiding illumination using real-time feedback of blood flow dynamics also minimized unwanted photodamage to the vascular network. Finally, through quantifiable behavior deficits and chronic imaging we show that this model can be used to study recovery mechanisms or the effects of therapeutics longitudinally.R01 EB021018 - NIBIB NIH HHS; R01 MH111359 - NIMH NIH HHS; R01 NS108472 - NINDS NIH HHSPublished versio

PEG-Like Nanoprobes: Multimodal, Pharmacokinetically and Optically Tunable Nanomaterials

“PEG-like Nanoprobes” (PN’s) are pharmacokinetically and optically tunable nanomaterials whose disposition in biological systems can be determined by fluorescence or radioactivity. PN’s feature a unique design where a single PEG polymer surrounds a short fluorochrome and radiometal bearing peptide, and endows the resulting nanoprobe with pharmacokinetic control (based on molecular weight of the PEG selected) and optical tunability (based on the fluorochrome selected), while the chelate provides a radiolabeling option. PN’s were used to image brain capillary angiography (intravital 2-photon microscopy), tumor capillary permeability (intravital fluorescent microscopy), and the tumor enhanced permeability and retention (EPR) effect (111In-PN and SPECT). Clinical applications of PN’s include use as long blood half-life fluorochromes for intraoperative angiography, for measurements of capillary permeability in breast cancer lesions, and to image EPR by SPECT, for stratifying patient candidates for long-circulating nanomedicines that may utilize the EPR mechanism

More Homogeneous Capillary Flow and Oxygenation in Deeper Cortical Layers Correlate with Increased Oxygen Extraction

Our understanding of how capillary blood flow and oxygen distribute across cortical layers to meet the local metabolic demand is incomplete. We addressed this question by using two-photon imaging of resting-state microvascular oxygen partial pressure (PO2) and flow in the whisker barrel cortex in awake mice. Our measurements in layers I-V show that the capillary red-blood-cell flux and oxygenation heterogeneity, and the intracapillary resistance to oxygen delivery, all decrease with depth, reaching a minimum around layer IV, while the depth-dependent oxygen extraction fraction is increased in layer IV, where oxygen demand is presumably the highest. Our findings suggest that more homogeneous distribution of the physiological observables relevant to oxygen transport to tissue is an important part of the microvascular network adaptation to local brain metabolism. These results will inform the biophysical models of layer-specific cerebral oxygen delivery and consumption and improve our understanding of the diseases that affect cerebral microcirculation

Differential effects of anesthetics on resting state functional connectivity in the mouse

Blood oxygen level-dependent (BOLD) functional MRI (fMRI) is a standard approach to examine resting state functional connectivity (RSFC), but fMRI in animal models is challenging. Recently, functional optical intrinsic signal imaging-which relies on the same hemodynamic signal underlying BOLD fMRI-has been developed as a complementary approach to assess RSFC in mice. Since it is difficult to ensure that an animal is in a truly resting state while awake, RSFC measurements under anesthesia remain an important approach. Therefore, we systematically examined measures of RSFC using non-invasive, widefield optical intrinsic signal imaging under five different anesthetics in male C57BL/6J mice. We find excellent seed-based, global, and interhemispheric connectivity using tribromoethanol (Avertin) and ketamine-xylazine, comparable to results in the literature including awake animals. Urethane anesthesia yielded intermediate results, while chloral hydrate and isoflurane were both associated with poor RSFC. Furthermore, we found a correspondence between the strength of RSFC and the power of low-frequency hemodynamic fluctuations. In conclusion, Avertin and ketamine-xylazine provide robust and reproducible measures of RSFC in mice, whereas chloral hydrate and isoflurane do not.R25 NS065743 - NINDS NIH HHS; KL2 TR002542 - NCATS NIH HHS; R00 AG042026 - NIA NIH HHS; K08 NS112601 - NINDS NIH HHS; P01 NS055104 - NINDS NIH HHS; R01 MH111359 - NIMH NIH HHS; R01 NS102969 - NINDS NIH HHS; R01 AA027097 - NIAAA NIH HHS; R01 NS091230 - NINDS NIH HHSPublished versio

Intrinsic optical signal imaging of the blood volume changes is sufficient for mapping the resting state functional connectivity in the rodent cortex

Published in final edited form as: J Neural Eng. 2018 June 01; 15(3): 035003–. doi:10.1088/1741-2552/aaafe4.Objective. Resting state functional connectivity (RSFC) allows the study of functional organization in normal and diseased brain by measuring the spontaneous brain activity generated under resting conditions. Intrinsic optical signal imaging (IOSI) based on multiple illumination wavelengths has been used successfully to compute RSFC maps in animal studies. The IOSI setup complexity would be greatly reduced if only a single wavelength can be used to obtain comparable RSFC maps. Approach. We used anesthetized mice and performed various comparisons between the RSFC maps based on single wavelength as well as oxy-, deoxy- and total hemoglobin concentration changes. Main results. The RSFC maps based on IOSI at a single wavelength selected for sensitivity to the blood volume changes are quantitatively comparable to the RSFC maps based on oxy- and total hemoglobin concentration changes obtained by the more complex IOSI setups. Moreover, RSFC maps do not require CCD cameras with very high frame acquisition rates, since our results demonstrate that they can be computed from the data obtained at frame rates as low as 5 Hz. Significance. Our results will have general utility for guiding future RSFC studies based on IOSI and making decisions about the IOSI system designs.We are grateful to Adam Bauer for his guidance on replicating their experimental setup, and Silvina Ferradal and Erin Buckley for useful discussions. We gratefully acknowledge support from the NIH grants NS091230, NS055104, NS057198, EB021018, EB00790, and EB018464, the Fondation Leducq, the State Scholarship Fund of the China Scholarship Council (Construction of high-level university projects, No. 201406100123), and the Natural Science Foundation of China (NSFC, nos. 81472150). (NS091230 - NIH; NS055104 - NIH; NS057198 - NIH; EB021018 - NIH; EB00790 - NIH; EB018464 - NIH; Fondation Leducq; 201406100123 - China Scholarship Council; 81472150 - Natural Science Foundation of China (NSFC))https://iopscience.iop.org/article/10.1088/1741-2552/aaafe4/metaAccepted manuscrip

Neurovascular coupling is preserved in chronic stroke recovery after targeted photothrombosis

Functional neuroimaging, which measures hemodynamic responses to brain activity, has great potential for monitoring recovery in stroke patients and guiding rehabilitation during recovery. However, hemodynamic responses after stroke are almost always altered relative to responses in healthy subjects and it is still unclear if these alterations reflect the underlying brain physiology or if the alterations are purely due to vascular injury. In other words, we do not know the effect of stroke on neurovascular coupling and are therefore limited in our ability to use functional neuroimaging to accurately interpret stroke pathophysiology. To address this challenge, we simultaneously captured neural activity, through fluorescence calcium imaging, and hemodynamics, through intrinsic optical signal imaging, during longitudinal stroke recovery. Our data suggest that neurovascular coupling was preserved in the chronic phase of recovery (2 weeks and 4 weeks post-stoke) and resembled pre-stroke neurovascular coupling. This indicates that functional neuroimaging faithfully represents the underlying neural activity in chronic stroke. Further, neurovascular coupling in the sub-acute phase of stroke recovery was predictive of long-term behavioral outcomes. Stroke also resulted in increases in global brain oscillations, which showed distinct patterns between neural activity and hemodynamics. Increased neural excitability in the contralesional hemisphere was associated with increased contralesional intrahemispheric connectivity. Additionally, sub-acute increases in hemodynamic oscillations were associated with improved sensorimotor outcomes. Collectively, these results support the use of hemodynamic measures of brain activity post-stroke for predicting functional and behavioral outcomes

Development of [¹²³I]IPEB and [¹²³I]IMPEB as SPECT Radioligands for Metabotropic Glutamate Receptor Subtype 5

mGlu₅ play an important role in physiology and pathology to various central nervous system (CNS) diseases. Several positron emission tomography (PET) radiotracers have been developed to explore the role of mGlu₅ in brain disorders. However, there are no single photon emission computed tomography (SPECT) radioligands for mGlu₅. Here we report development of [¹²³I]­IPEB ([¹²³I]<b>1</b>) and [¹²³I]­IMPEB ([¹²³I]<b>2</b>) as mGlu₅ radioligands for SPECT. [¹²³I]<b>1</b> and [¹²³I]<b>2</b> were produced by copper­(I) mediated aromatic halide displacement reactions. The SPECT imaging using mouse models demonstrated that [¹²³I]<b>1</b> readily entered the brain and accumulated specifically in mGlu₅-rich regions of the brain such as striatum and hippocampus. However, in comparison to the corresponding PET tracer [¹⁸F]­FPEB, [¹²³I]<b>1</b> showed faster washout from the brain. The binding ratios of the striatum and the hippocampus compared to the cerebellum for [¹²³I]<b>1</b> and [¹⁸F]­FPEB were similar despite unfavorable pharmacokinetics of [¹²³I]<b>1</b>. Further structural optimization of <b>1</b> may lead to more viable SPECT radiotracers for the imaging of mGlu₅

Anatomical Modeling of Brain Vasculature in Two-Photon Microscopy by Generalizable Deep Learning

Objective and Impact Statement. Segmentation of blood vessels from two-photon microscopy (2PM) angiograms of brains has important applications in hemodynamic analysis and disease diagnosis. Here, we develop a generalizable deep learning technique for accurate 2PM vascular segmentation of sizable regions in mouse brains acquired from multiple 2PM setups. The technique is computationally efficient, thus ideal for large-scale neurovascular analysis. Introduction. Vascular segmentation from 2PM angiograms is an important first step in hemodynamic modeling of brain vasculature. Existing segmentation methods based on deep learning either lack the ability to generalize to data from different imaging systems or are computationally infeasible for large-scale angiograms. In this work, we overcome both these limitations by a method that is generalizable to various imaging systems and is able to segment large-scale angiograms. Methods. We employ a computationally efficient deep learning framework with a loss function that incorporates a balanced binary-cross-entropy loss and total variation regularization on the network’s output. Its effectiveness is demonstrated on experimentally acquired in vivo angiograms from mouse brains of dimensions up to 808×808×702 μm. Results. To demonstrate the superior generalizability of our framework, we train on data from only one 2PM microscope and demonstrate high-quality segmentation on data from a different microscope without any network tuning. Overall, our method demonstrates 10× faster computation in terms of voxels-segmented-per-second and 3× larger depth compared to the state-of-the-art. Conclusion. Our work provides a generalizable and computationally efficient anatomical modeling framework for brain vasculature, which consists of deep learning-based vascular segmentation followed by graphing. It paves the way for future modeling and analysis of hemodynamic response at much greater scales that were inaccessible before

Dynamic capillary stalls in reperfused ischemic penumbra contribute to injury:A hyperacute role for neutrophils in persistent traffic jams

Ever since the introduction of thrombolysis and the subsequent expansion of endovascular treatments for acute ischemic stroke, it remains to be identified why the actual outcomes are less favorable despite recanalization. Here, by high spatio-temporal resolution imaging of capillary circulation in mice, we introduce the pathological phenomenon of dynamic flow stalls in cerebral capillaries, occurring persistently in salvageable penumbra after reperfusion. These stalls, which are different from permanent cellular plugs of no-reflow, were temporarily and repetitively occurring in the capillary network, impairing the overall circulation like small focal traffic jams. In vivo microscopy in the ischemic penumbra revealed leukocytes traveling slowly through capillary lumen or getting stuck, while red blood cell flow was being disturbed in the neighboring segments under reperfused conditions. Stall dynamics could be modulated, by injection of an anti-Ly6G antibody specifically targeting neutrophils. Decreased number and duration of stalls were associated with improvement in penumbral blood flow within 2–24 h after reperfusion along with increased capillary oxygenation, decreased cellular damage and improved functional outcome. Thereby, dynamic microcirculatory stall phenomenon can be a contributing factor to ongoing penumbral injury and is a potential hyperacute mechanism adding on previous observations of detrimental effects of activated neutrophils in ischemic stroke