Abstract P434: A Mouse Model To Control Vessel Diameter With Light

2021 ◽  
Vol 129 (Suppl_1) ◽  
Author(s):  
Philip O'Herron ◽  
David Hartmann ◽  
Andy Y Shih
Keyword(s):  
Smooth Muscle ◽  
Cognitive Impairment ◽  
Vascular Function ◽  
Large Scale ◽  
Imaging System ◽  
Contractile Function ◽  
Small Individual ◽  
Two Photon ◽  
Cranial Window

In diseases such as stroke, hypertension, vascular cognitive impairment, and Alzheimer’s disease, defects in the cerebrovascular system lead to reduced blood flow and vasoreactivity to stimuli. Recently, there has been increased appreciation for the role of small vessels in these vascular pathologies. For example, small vessel dysfunction can cause widespread microinfarcts and capillary stalling, which may underlie cognitive impairment in cases where large scale vascular abnormalities are not readily detected. However, vascular function is difficult to dissociate from concurrent neuronal deficits cause by damage to neuronal circuitry in brain pathology. Thus the ability to directly probe smooth muscle contraction of small, individual vessels in the intact brain would be a valuable tool for increasing our understanding of vascular contributions to cognitive impairment. We developed an experimental paradigm to optically probe the contractile function of arterioles in vivo with high spatiotemporal precision. This was done by expressing the excitatory opsin ReaChR in vascular smooth muscle cells and pericytes. Using a 594 nm light-emitting diode we were able to evoke widespread vasoconstriction across the cranial window. With a 1040 nm focused, pulsed laser for two-photon stimulation, we were able to evoke highly localized constrictions targeted to individual pial artery branches or penetrating arterioles. Our dual light-path imaging system allowed the optogenetic stimulation to be performed with simultaneous two-photon imaging to monitor vessel activity. Using a spatial light modulator, we were also able to constrict vessels both above and below the imaging plane. This is a powerful tool to assay vasoconstrictive function of single arterioles across 3-dimensional vascular networks in vivo. It also presents novel opportunities to study conductance of vascular signals and to modulate dynamics of functional hyperemia.

scholarly journals A method for noninvasive longitudinal measurements of [Ca2+] in arterioles of hypertensive optical biosensor mice

2014 ◽  
Vol 307 (2) ◽  
pp. H173-H181 ◽  
Author(s):  
Joseph R. H. Mauban ◽  
Seth T. Fairfax ◽  
Mark A. Rizzo ◽  
Jin Zhang ◽  
Withrow Gil Wier
Keyword(s):  
Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Vascular Function ◽  
Resonance Energy ◽  
Extended Period ◽  
Muscle Cells ◽  
Optical Biosensor ◽  
Ang Ii ◽  
Two Photon

We used two-photon (2-p) Förster resonance energy transfer (FRET) microscopy to provide serial, noninvasive measurements of [Ca2+] in arterioles of living “biosensor” mice. These express a genetically encoded Ca2+ indicator (GECI), either FRET-based exMLCK or intensity-based GCaMP2. The FRET ratios, Rmin and Rmax, required for in vivo Ca2+ calibration of exMLCK were obtained in isolated arteries. For in vivo experiments, mice were anesthetized (1.5% isoflurane), and arterioles within a depilated ear were visualized through the intact skin (i.e., noninvasively), by 2-p excitation of exMLCK (at 820 nm) or GCaMP2 (at 920 nm). Spontaneous or agonist-evoked [Ca2+] transients in arteriolar smooth muscle cells were imaged (at 2 Hz) with both exMLCK and GCaMP2. To examine changes in arteriolar [Ca2+] that might accompany hypertension, five exMLCK mice were implanted with telemetric blood pressure transducers and osmotic minipumps containing ANG II (350 ng·kg−1·min−1) and fed a high (6%)-salt diet for 9 days. [Ca2+] was measured every other day in five smooth muscle cells of two to three arterioles in each animal. Prior to ANG II/salt, [Ca2+] was 246 ± 42 nM. [Ca2+] increased transiently to 599 nM on day 2 after beginning ANG II/salt, then remained elevated at 331 ± 42 nM for 4 more days, before returning to 265 ± 47 nM 6 days after removal of ANG II/salt. In summary, two-photon excitation of exMLCK and GCaMP2 provides a method for noninvasive, longitudinal quantification of [Ca2+] dynamics and vascular structure in individual arterioles of a particular animal over an extended period of time, a capability that should enhance future studies of hypertension and vascular function.

scholarly journals Fundamental Roles of Axial Stretch in Isometric and Isobaric Evaluations of Vascular Contractility

2019 ◽  
Vol 141 (3) ◽  
Author(s):  
Alexander W. Caulk ◽  
Jay D. Humphrey ◽  
Sae-Il Murtada
Keyword(s):  
Smooth Muscle ◽  
Vascular Smooth Muscle ◽  
Muscle Function ◽  
Contractile Function ◽  
Contractile Activity ◽  
Comparison Of Methods ◽  
Axial Stretch ◽  
Smooth Muscle Function ◽  
In Vivo Loading

Vascular smooth muscle cells (VSMCs) can regulate arterial mechanics via contractile activity in response to changing mechanical and chemical signals. Contractility is traditionally evaluated via uniaxial isometric testing of isolated rings despite the in vivo environment being very different. Most blood vessels maintain a locally preferred value of in vivo axial stretch while subjected to changes in distending pressure, but both of these phenomena are obscured in uniaxial isometric testing. Few studies have rigorously analyzed the role of in vivo loading conditions in smooth muscle function. Thus, we evaluated effects of uniaxial versus biaxial deformations on smooth muscle contractility by stimulating two regions of the mouse aorta with different vasoconstrictors using one of three testing protocols: (i) uniaxial isometric testing, (ii) biaxial isometric testing, and (iii) axially isometric plus isobaric testing. Comparison of methods (i) and (ii) revealed increased sensitivity and contractile capacity to potassium chloride and phenylephrine (PE) with biaxial isometric testing, and comparison of methods (ii) and (iii) revealed a further increase in contractile capacity with isometric plus isobaric testing. Importantly, regional differences in estimated in vivo axial stretch suggest locally distinct optimal biaxial configurations for achieving maximal smooth muscle contraction, which can only be revealed with biaxial testing. Such differences highlight the importance of considering in vivo loading and geometric configurations when evaluating smooth muscle function. Given the physiologic relevance of axial extension and luminal pressurization, we submit that, when possible, axially isometric plus isobaric testing should be employed to evaluate vascular smooth muscle contractile function.

scholarly journals Fast and robust active neuron segmentation in two-photon calcium imaging using spatiotemporal deep learning

2019 ◽  
Vol 116 (17) ◽  
pp. 8554-8563 ◽  
Author(s):  
Somayyeh Soltanian-Zadeh ◽  
Kaan Sahingur ◽  
Sarah Blau ◽  
Yiyang Gong ◽  
Sina Farsiu
Keyword(s):  
Real Time ◽  
Calcium Imaging ◽  
Large Scale ◽  
Cortical Layer ◽  
Fast Method ◽  
Active Neuron ◽  
Neuronal Coding ◽  
Two Photon ◽  
Neuron Density

Calcium imaging records large-scale neuronal activity with cellular resolution in vivo. Automated, fast, and reliable active neuron segmentation is a critical step in the analysis workflow of utilizing neuronal signals in real-time behavioral studies for discovery of neuronal coding properties. Here, to exploit the full spatiotemporal information in two-photon calcium imaging movies, we propose a 3D convolutional neural network to identify and segment active neurons. By utilizing a variety of two-photon microscopy datasets, we show that our method outperforms state-of-the-art techniques and is on a par with manual segmentation. Furthermore, we demonstrate that the network trained on data recorded at a specific cortical layer can be used to accurately segment active neurons from another layer with different neuron density. Finally, our work documents significant tabulation flaws in one of the most cited and active online scientific challenges in neuron segmentation. As our computationally fast method is an invaluable tool for a large spectrum of real-time optogenetic experiments, we have made our open-source software and carefully annotated dataset freely available online.

scholarly journals Sympathetic nerve-dependent regulation of mucosal vascular tone modifies airway smooth muscle reactivity

2010 ◽  
Vol 109 (5) ◽  
pp. 1292-1300 ◽  
Author(s):  
Stuart B. Mazzone ◽  
Lina H. K. Lim ◽  
Elizabeth M. Wagner ◽  
Nanako Mori ◽  
Brendan J. Canning
Keyword(s):  
Blood Flow ◽  
Smooth Muscle ◽  
Airway Smooth Muscle ◽  
Vascular Function ◽  
Adrenergic Receptor ◽  
Muscle Tone ◽  
Mucosal Blood Flow ◽  
Nerve Fibers ◽  
Neurokinin A

The airways contain a dense subepithelial microvascular plexus that is involved in the supply and clearance of substances to and from the airway wall. We set out to test the hypothesis that airway smooth muscle reactivity to bronchoconstricting agents may be dependent on airway mucosal blood flow. Immunohistochemical staining identified vasoconstrictor and vasodilator nerve fibers associated with subepithelial blood vessels in the guinea pig airways. Intravital microscopy of the tracheal mucosal microvasculature in anesthetized guinea pigs revealed that blockade of α-adrenergic receptors increased baseline arteriole diameter by ∼40%, whereas the α-adrenergic receptor agonist phenylephrine produced a modest (5%) vasoconstriction in excess of the baseline tone. In subsequent in vivo experiments, tracheal contractions evoked by topically applied histamine were significantly reduced ( P < 0.05) and enhanced by α-adrenergic receptor blockade and activation, respectively. α-Adrenergic ligands produced similar significant ( P < 0.05) effects on airway smooth muscle contractions evoked by topically administered capsaicin, intravenously administered neurokinin A, inhaled histamine, and topically administered antigen in sensitized animals. These responses were independent of any direct effect of α-adrenergic ligands on the airway smooth muscle tone. The data suggest that changes in blood flow in the vessels supplying the airways regulate the reactivity of the underlying airway smooth muscle to locally released and exogenously administered agents by regulating their clearance. We speculate that changes in mucosal vascular function or changes in neuronal regulation of the airway vasculature may contribute to airways responsiveness in disease.

scholarly journals Segmentation-less, automated vascular vectorization robustly extracts neurovascular network statistics from in vivo two-photon images

2020 ◽  
Author(s):  
Samuel A. Mihelic ◽  
William A. Sikora ◽  
Ahmed M. Hassan ◽  
Michael R. Williamson ◽  
Theresa A. Jones ◽  
...  
Keyword(s):  
Image Segmentation ◽  
Statistical Power ◽  
Large Scale ◽  
Performance Metrics ◽  
Three Dimensional ◽  
Vascular Anatomy ◽  
Input Image ◽  
Network Graph ◽  
Two Photon

AbstractRecent advances in two-photon microscopy (2PM) have allowed large scale imaging and analysis of cortical blood vessel networks in living mice. However, extracting a network graph and vector representations for vessels remain bottlenecks in many applications. Vascular vectorization is algorithmically difficult because blood vessels have many shapes and sizes, the samples are often unevenly illuminated, and large image volumes are required to achieve good statistical power. State-of-the-art, three-dimensional, vascular vectorization approaches require a segmented/binary image, relying on manual or supervised-machine annotation. Therefore, voxel-by-voxel image segmentation is biased by the human annotator/trainer. Furthermore, segmented images oftentimes require remedial morphological filtering before skeletonization/vectorization. To address these limitations, we propose a vectorization method to extract vascular objects directly from unsegmented images. The Segmentation-Less, Automated, Vascular Vectorization (SLAVV) source code in MATLAB is openly available on GitHub. This novel method uses simple models of vascular anatomy, efficient linear filtering, and low-complexity vector extraction algorithms to remove the image segmentation requirement, replacing it with manual or automated vector classification. SLAVV is demonstrated on three in vivo 2PM image volumes of microvascular networks (capillaries, arterioles and venules) in the mouse cortex. Vectorization performance is proven robust to the choice of plasma- or endothelial-labeled contrast, and processing costs are shown to scale with input image volume. Fully-automated SLAVV performance is evaluated on various, simulated 2PM images based on the large, [1.4, 0.9, 0.6] mm input image, and performance metrics show greater robustness to image quality than an intensity-based thresholding approach.

Changes in Vascular Viscoelasticity Caused by Smooth Muscle Cell Tone and Pressurization Frequency

2007 ◽  
Author(s):  
Ryan J. DeWall ◽  
Naomi C. Chesler
Keyword(s):  
Smooth Muscle ◽  
Energy Dissipation ◽  
Smooth Muscle Cell ◽  
Muscle Cell ◽  
Vascular Function ◽  
Viscoelastic Properties ◽  
Carotid Arteries ◽  
The Body ◽  
Thromboxane Receptor

The cushioning and conduit functions of large arteries allow the body to efficiently circulate blood and perfuse organs. These functions can be characterized by quantifying the viscoelastic properties of arteries. In this study, we investigated the effects of smooth muscle cell (SMC) tone and pressurization frequency on vascular viscoelasticity using an isolated, perfused vessel test. We tested mouse carotid arteries in control, dilated (via sodium nitroprusside) and constricted (via the thromboxane receptor analogue U46619) states at 0.1, 1, 3, and 5 Hz with a pulse pressure from 90 to 120 mmHg. Dose response experiments were first performed in order to determine the optimal vasoconstrictor concentration to be used in frequency response experiments. Our results showed that energy dissipation was significantly higher and elasticity was significantly lower for vasoconstricted arteries. We also found that frequency significantly changed both energy dissipation and elasticity as the frequency was increased from 0.1 to 5 Hz. These results provide insights into the changes in vascular viscoelasticity caused by SMC tone and pressurization frequency, which have implications for vascular function in vivo.

Exposure of immature rats to hyperoxia increases tracheal smooth muscle stress generation in vitro

1994 ◽  
Vol 76 (2) ◽  
pp. 743-749 ◽  
Author(s):  
M. B. Hershenson ◽  
M. E. Wylam ◽  
N. Punjabi ◽  
J. G. Umans ◽  
P. T. Schumacker ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Contractile Function ◽  
Stress Generation ◽  
Tracheal Smooth Muscle ◽  
Smooth Muscle Contractility ◽  
Cross Sectional ◽  
Induced Stress ◽  
Hyperoxic Exposure

Recently, we demonstrated that chronic exposure to hyperoxia causes in vivo airway muscarinic receptor hyperresponsiveness in the developing rat [Am. J. Physiol. 262 (Lung Cell. Mol. Physiol. 6): L263-L269, 1992]. To test whether airway cholinergic hyperresponsiveness might result from intrinsic alterations in smooth muscle contractility, we measured the effect of in vivo hyperoxia on the contractile force elicited by acetylcholine (ACh) of isometrically mounted tracheal rings in vitro. Tracheal rings were obtained from 3-wk-old rats exposed to air or to > 95% O2 for 8 days. Muscarinic responses were determined by measuring the force elicited by exposure to increasing concentrations of ACh. Responses were normalized to the morphometrically determined tracheal smooth muscle cross-sectional area in a plane perpendicular to the axis of force generation. In vivo O2 exposure significantly increased maximal ACh-induced stress generation (response to 10(-3) M ACh: air, 15.92 +/- 1.37 g/mm2; O2, 21.78 +/- 1.52 g/mm2; P = 0.010). The ACh-induced stress generation of cylinders from hyperoxic rats was substantially reduced by both epithelial removal and treatment with the cyclooxygenase inhibitor indomethacin. We conclude that in vivo hyperoxic exposure increases tracheal smooth muscle contractile function in vitro and that epithelium-derived prostaglandin(s) contributes to the observed increase in maximal contractile responsiveness.

Phospholipase C Isoforms, Cytoskeletal Organization, and Vascular Smooth Muscle Differentiation

Physiology ◽  
2000 ◽  
Vol 15 (1) ◽  
pp. 41-45 ◽  
Author(s):  
Joanne S. Lymn ◽  
Alun D. Hughes
Keyword(s):  
Smooth Muscle ◽  
Vascular Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Phospholipase C ◽  
Vascular Smooth Muscle Cells ◽  
Expression Profile ◽  
Contractile Function ◽  
Muscle Cells ◽  
Cytoskeletal Organization

The function of differentiated vascular smooth muscle cells (VSMC) in vivo is the regulation of contractility. Following injury or disease, however, VSMC lose their contractile function and take on a synthetic, proliferative phenotype. This dedifferentiation is generally accompanied by a change in the expression profile of phospholipase C isoforms.

scholarly journals Role of Pial Microvasospasms and Leukocyte Plugging for Parenchymal Perfusion after Subarachnoid Hemorrhage Assessed by In Vivo Multi-Photon Microscopy

2021 ◽  
Vol 22 (16) ◽  
pp. 8444
Author(s):  
Julian Schwarting ◽  
Kathrin Nehrkorn ◽  
Hanhan Liu ◽  
Nikolaus Plesnila ◽  
Nicole Angela Terpolilli
Keyword(s):  
Subarachnoid Hemorrhage ◽  
Cerebral Ischemia ◽  
Fluorescent Dyes ◽  
Delayed Cerebral Ischemia ◽  
Tissue Imaging ◽  
Two Photon Microscopy ◽  
Two Photon ◽  
Cranial Window ◽  
Pial Arterioles

Subarachnoid hemorrhage (SAH) is associated with acute and delayed cerebral ischemia. We suggested spasms of pial arterioles as a possible mechanism; however, it remained unclear whether and how pial microvasospasms (MVSs) induce cerebral ischemia. Therefore, we used in vivo deep tissue imaging by two-photon microscopy to investigate MVSs together with the intraparenchymal microcirculation in a clinically relevant murine SAH model. Male C57BL/6 mice received a cranial window. Cerebral vessels and leukocytes were labelled with fluorescent dyes and imaged by in vivo two-photon microscopy before and three hours after SAH induced by filament perforation. After SAH, a large clot formed around the perforation site at the skull base, and blood distributed along the perivascular space of the middle cerebral artery up to the cerebral cortex. Comparing the cerebral microvasculature before and after SAH, we identified three different patterns of constrictions: pearl string, global, and bottleneck. At the same time, the volume of perfused intraparenchymal vessels and blood flow velocity in individual arterioles were significantly reduced by more than 60%. Plugging of capillaries by leukocytes was observed but infrequent. The current study demonstrates that perivascular blood is associated with spasms of pial arterioles and that these spasms result in a significant reduction in cortical perfusion after SAH. Thus, the pial microvasospasm seems to be an important mechanism by which blood in the subarachnoid space triggers cerebral ischemia after SAH. Identifying the mechanisms of pial vasospasm may therefore result in novel therapeutic options for SAH patients.

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