Alzheimer’s disease (AD) is characterized by progressive, irreversible neurodegeneration, leading to memory loss and cognitive decline. In mouse models of AD, global decreases in cerebral blood flow (CBF) are brought on by the plugging of capillaries by arrested neutrophils, and the administration of the neutrophil-specific antibody against Ly6G (anti-Ly6G) reduces these capillary stalls in minutes and improves cognitive function within hours. This suggests that at least some aspects of neural activity impairment are reversible, but the mechanism of this recovery – and what specific neural activity is normalized – is not yet known. In agreement with prior studies, we found orientation tuning selectivity to drifting gratings in primary visual cortex neurons to be broadened in mouse models of AD. Here, we hypothesized that the impaired neural response can be modified by blood flow improvement with anti-Ly6G treatment (4 mg/kg) in the APP/PS1 mouse model of AD. We transfected neurons in layer 2/3 of the primary visual cortex (V1) of mice with a fluorescent calcium indicator using AAV9 vectors (pAAV.Syn.GCaMP6s.WPRE.SV40, 10^12 vg/mL). We also injected the fluorescent labels methoxy-X04 to detect amyloid plaques and Texas-red-dextran in the vasculature to detect blocked capillaries. Drifting grating visual stimuli were presented to anesthetized mice with using MouseGoggles during recording with two-photon microscopy before and one day after anti-Ly6G or isotype control antibody administration (Fig. 1). One day after anti-Ly6G administration to reduce stalls and increase CBF, we observed a significant sharpening of orientation tuning relative to baseline in the same animals, as well as a trend toward increased spontaneous activity in V1 neurons (Fig. 2). Such normalization of neural activity patterns likely underlies the improved cognitive function that is seen within hours of blood flow increase in AD mouse models. Our data suggests some aspects of the neural and behavioral deficits in AD are acutely recoverable by increasing CBF. Such recovery demonstrates a promising avenue for future therapeutic targets to combat the symptoms of AD in humans.

Several genetic and cardiovascular risk factors increase incidence of Alzheimer’s disease and related dementias (ADRD). Hypertension and the ε4 allele of apolipoprotein E (ApoE) are powerful drivers of cognitive impairment in ADRD. These risk factors are also associated with decreased cerebral blood flow (CBF). Experimental data suggest that the CBF reduction may be due to temporary capillary occlusions (capillary stalls) caused by circulating vascular and immune cells. To gain insight into how these risk factors may contribute to cognitive decline, we induced hypertension in mice expressing human ApoE3 or E4 (ApoE3- or E4-TR mice), and focused on the resulting CBF and cognitive alterations, and their potential reversal by preventing capillary stalling. Male ApoE3 and 4-TR mice (n = 9-15/group) were assessed at three timepoints: Baseline, following two weeks of angiotensin II-induced hypertension (AngII, 500ng/kg/min), and after an additional week of anti-platelet treatment (prasugrel, 10mg/kg, daily). We tested spatial memory function in a Y-maze, and imaged cortical microvascular flow using two-photon microscopy. At baseline, ApoE3 and 4-TR mice showed similar memory performance, but with hypertension (∼25 mmHg increase in blood pressure) ApoE4-TR mice exhibited a spatial working memory deficit, which coincided with a 26% decrease in capillary flow speed (4.11±0.3 vs. 3.05±0.29 mm/s, p <0.01), and a 1.8X increase in the incidence of non-flowing capillaries. Most stalled capillaries contained only red blood cells in the stalled segment (57%), with some also containing leukocytes (30%), or platelets (12%) (Fig. 1). Treatment with prasugrel led to a 55% reduction in stalling (65±9 vs. 144±24 stalls/mm 3 ), and a concomitant 23% increase in capillary blood flow (3.75±0.36 vs. 3.05±0.29 mm/s, p = 0.11), and significantly improved blood flow in penetrating arterioles that correlated with restored performance on the Y-maze task (Fig. 2). Immunohistochemistry further revealed improved BBB integrity and reduced gliosis following prasugrel treatment. Paralleling human findings, we show that mice with the e4 allele of ApoE experience more deleterious consequences from hypertension, as compared to their e3 counterparts. Prasugrel treatment led to an almost complete recovery of these deficits, suggesting that anti-platelet agents may be of therapeutic value by improving CBF and cognitive function in the presence of these risk factors.

Third harmonic generation with 1320-nm, femtosecond pulses can visualize individual myelinated axons in subcortical white matter through intact cortex of live mice. In a cuprizone multiple sclerosis model, this enabled longitudinal tracking of myelin damage.

INTRODUCTION:In this study, we explore the role of oxidative stress produced by NOX2-containing NADPH oxidase as a molecular mechanism causing capillary stalling and cerebral blood flow deficits in the APP/PS1 mouse model of AD. METHODS:We inhibited NOX2 in APP/PS1 mice by administering a 10 mg/kg dose of the peptide inhibitor gp91-ds-tat i.p., for two weeks. We used in vivo two-photon imaging to measure capillary stalling, penetrating arteriole flow, and vascular inflammation. We also characterized short-term memory function and gene expression changes in cerebral microvessels. RESULTS:We found that after NOX2 inhibition capillary stalling, as well as parenchymal and vascular inflammation, were significantly reduced. In addition, we found a significant increase in penetrating arteriole flow, followed by an improvement in short-term memory, and downregulation of inflammatory gene expression pathways. DISCUSSION:Oxidative stress is a major mechanism leading to microvascular dysfunction in AD, and represents an important therapeutic target.

Optogenetic effectors and sensors provide a novel real-time window into complex physiological processes, enabling determination of molecular signaling processes within functioning cellular networks. However, the combination of these optical tools in mice is made practical by construction of genetic lines that are optically compatible and genetically tractable. We present a new toolbox of 21 mouse lines with lineage-specific expression of optogenetic effectors and sensors for direct biallelic combination, avoiding the multiallelic requirement of Cre recombinase -mediated DNA recombination, focusing on models relevant for cardiovascular biology. Optogenetic effectors (11 lines) or Ca 2+ sensors (10 lines) were selectively expressed in cardiac pacemaker cells, cardiomyocytes, vascular endothelial and smooth muscle cells, alveolar epithelial cells, lymphocytes, glia, and other cell types. Optogenetic effector and sensor function was demonstrated in numerous tissues. Arterial/arteriolar tone was modulated by optical activation of the second messengers InsP 3 (optoα1AR) and cAMP (optoß2AR), or Ca 2+ -permeant membrane channels (CatCh2) in smooth muscle ( Acta2 ) and endothelium ( Cdh5 ). Cardiac activation was separately controlled through activation of nodal/conducting cells or cardiac myocytes. We demonstrate combined effector and sensor function in biallelic mouse crosses: optical cardiac pacing and simultaneous cardiomyocyte Ca 2+ imaging in Hcn4 BAC -CatCh2/ Myh6 -GCaMP8 crosses. These experiments highlight the potential of these mice to explore cellular signaling in vivo, in complex tissue networks.

BackgroundThe study of functional cardiomyocyte adaptation and inflammatory cell behavior at the micro‐scale in vivo has been challenging due to limited imaging tools. We recently developed intravital multiphoton microscopy (MPM) methods that enable visualization and quantification of cardiac dynamics at the cell and micro‐vessel level throughout the cardiac cycle. We aimed to determine the dynamic cellular changes that occur due to high fat diet (HFD) induced hypertrophy using intravital cardiac MPM.MethodsApoE−/− C57Bl6 mice started a HFD at 6 weeks of age (ApoE−/−‐HFD, n=11), while age‐matched wild‐type mice were fed a normal chow diet (WT‐ND, n=10). At 26‐weeks, mice were assessed by cardiac echocardiography and intravital MPM in the intact beating heart. Intravenous injections of rhodamine‐6G (R6g) labeled cardiomyocytes and leukocytes, and Texas‐Red dextran labeled vasculature during intravital MPM. 3D volumes were reconstructed throughout the cardiac cycle to quantify cell motion using automated algorithms for cell displacement and regional deformation. Post‐mortem immuno‐histology were performed for myocardial macrophages (CD68), capillary density and cross‐sectional area (wheat‐germ‐agglutin).ResultsApoE−/−‐HFD hearts underwent hypertrophy compared to WT‐ND with increased heart weight‐to‐tibial length ratio (13±1.1 vs 10±0.8), left ventricle wall thickness (1.13±0.06 mm vs 1.07±0.03 mm) and myocyte cross‐sectional area (387±22.1 mm2 vs 278±16.6 mm2, p<0.05 for all), while ejection fraction remained preserved (59±3% vs 66±3%). Intravital MPM demonstrated that cardiomyocytes move a greater total distance during each cardiac cycle in ApoE−/−‐HFD vs WT‐ND. Maximum displacement in the apex‐base and anterior‐posterior directions increased by 56% in ApoE−/−‐HFD compared to WT‐ND (32±11 mm vs 18±6 mm), whereas regional absolute deformation remains similar between groups. R6g+ leukocytes were visible moving in capillaries. The incidence of patrolling behavior (defined as slow moving cells, visible for longer than one heartbeat) increased in capillaries of ApoE−/−‐HFD compared to WT‐ND (3.4±0.5/min vs 0.12±0.1/min, p<0.01), while the incidence of flowing (visible for less than one heartbeat) and non‐flowing (visible for 500 heartbeats) remained similar. Myocardial CD68+ macrophages increased (780±121/mm2 vs 89±20/mm2, p<0.0001) and capillary density decreased (3271±167/mm2 vs 3886±105/mm2, p=0.0067) in ApoE−/ −‐HFD hearts compared to WT‐ND in post‐mortem sections.ConclusionIntravital cardiac MPM provides a new perspective to study cardiac hypertrophy by capturing the simultaneous contributions of inflammatory cells and cardiomyocyte function in the beating heart. These results suggest that hypertrophied cardiomyocytes increase overall tissue motion to compensate for unchanged cardiomyocyte contraction to maintain a healthy ejection fraction. Increased capillary leukocyte patrolling behavior may promote myocardial hypertrophy.Support or Funding InformationAHA17POST33680127, NSFDBI1707312, NIH5R21EB02469403

BackgroundMultiphoton microscopy (MPM) has enabled in vivo time‐lapse imaging of the heart that shows motion of cells within the tissue with micrometer resolution. We developed automated analysis techniques to quantify cellular motion from in vivo cardiac MPM images throughout the cardiac cycle.MethodsIntravital cardiac MPM was performed on ventilated, male and female, 4–6 month‐old, C57Bl6 mice (n=9). MPM image volumes (100 μm deep) of the beating mouse left ventricle were acquired at 30 frames per second while recording the electrocardiogram and respiratory pressure (Fig. a). Image volumes were reconstructed by assembling lines acquired nearest to a specified point in the cardio‐respiratory phase space. Bulk‐tissue motion was calculated from the three‐dimensional translational transformation that best aligned images at each point of the cardiac cycle to a reference image at the most stable portion of the cardio‐respiratory phase space (respiratory 50%, cardiac 80%). After bulk‐tissue motion was subtracted, local tissue deformation was extracted via non‐rigid registration of the reconstructed images to the reference image and divergence was calculated from the resulting vector field. Analysis methods were applied to images before and after epinephrine injection (50 μg intramuscular), and following focal sterile injury by laser irradiation (FSI).ResultsVolumes were reconstructed in 50 intervals across the cardiac cycle that show vasculature (intravenous Texas‐red dextran, red) and cardiomyocytes and leukocytes (rhodamine 6G, cyan) moving across the field of view (Fig. b–c). Automated analysis of bulk‐tissue motion (Fig. d–e) indicated maximum displacements occurring at 16 % (anterior‐posterior direction), 36 % (superior‐inferior) and 38 % (epi‐endocardial) of the cardiac cycle. The calculated epi‐endocardial motion amplitude correlated with stroke volume (R2=0.35) measured by echocardiography in the same animal one day before MPM imaging. Automated analysis of local tissue deformation indicated a maximum absolute deformation occurring at 10 % of the cardiac cycle (Fig. f). Comparison to manual feature tracking across the cardiac cycle validated the automated measurements. Rigid and non‐rigid motion analysis techniques were applied to epinephrine stress test and focal sterile injury (FSI) lesion models showing, respectively, increased and decreased bulk‐tissue and local deformation and divergence.ConclusionsWe have shown and applied novel, fast, and accurate techniques for characterizing cardiac motion from in vivo cardiac MPM to study the performance of contractile cells in the heart during health and disease.Support or Funding InformationAmerican Heart Association (17POST33680127), National Institutes of Health (5R21EB024694‐03), and National Science Foundation (DBI‐1707312).Figure 1

Cardiovascular disease is the leading cause of worldwide mortality. Intravital microscopy has provided unprecedented insight into leukocyte biology by enabling the visualization of dynamic responses within living organ systems at the cell-scale. The heart presents a uniquely dynamic microenvironment driven by periodic, synchronous electrical conduction leading to rhythmic contractions of cardiomyocytes, and phasic coronary blood flow. In addition to functions shared throughout the body, immune cells have specific functions in the heart including tissue-resident macrophage-facilitated electrical conduction and rapid monocyte infiltration upon injury. Leukocyte responses to cardiac pathologies, including myocardial infarction and heart failure, have been well-studied using standard techniques, however, certain questions related to spatiotemporal relationships remain unanswered. Intravital imaging techniques could greatly benefit our understanding of the complexities of in vivo leukocyte behavior within cardiac tissue, but these techniques have been challenging to apply. Different approaches have been developed including high frame rate imaging of the beating heart, explantation models, micro-endoscopy, and mechanical stabilization coupled with various acquisition schemes to overcome challenges specific to the heart. The field of cardiac science has only begun to benefit from intravital microscopy techniques. The current focused review presents an overview of leukocyte responses in the heart, recent developments in intravital microscopy for the murine heart, and a discussion of future developments and applications for cardiovascular immunology.

Cardiovascular disease is the leading cause of worldwide mortality. Intravital microscopy has provided unprecedented insight into leukocyte biology by enabling the visualization of dynamic responses within living organ systems at the cell-scale. The heart presents a uniquely dynamic microenvironment driven by periodic, synchronous electrical conduction leading to rhythmic contractions of cardiomyocytes, and phasic coronary blood flow. In addition to functions shared throughout the body, immune cells have specific functions in the heart including tissue-resident macrophage-facilitated electrical conduction and rapid monocyte infiltration upon injury. Leukocyte responses to cardiac pathologies, including myocardial infarction and heart failure, have been well-studied using standard techniques, however, certain questions related to spatiotemporal relationships remain unanswered. Intravital imaging techniques could greatly benefit our understanding of the complexities of in vivo leukocyte behavior within cardiac tissue, but these techniques have been challenging to apply. Different approaches have been developed including high frame rate imaging of the beating heart, explantation models, micro-endoscopy, and mechanical stabilization coupled with various acquisition schemes to overcome challenges specific to the heart. The field of cardiac science has only begun to benefit from intravital microscopy techniques. The current focused review presents an overview of leukocyte responses in the heart, recent developments in intravital microscopy for the murine heart, and a discussion of future developments and applications for cardiovascular immunology.

Optical coherence microscopy (OCM) provides non-invasive, label-free, cellular-resolution imaging based on optical scattering contrast. Its interferometric detection captures the optical field, providing opportunities for computational reconstruction. However, the depth coverage of OCM is restricted by defocus and photon collection, and its penetration depth is limited by multiple scattering (MS). Here, we propose integrating hardware and computational adaptive optics in different ways, to improve the throughput, penetration depth, and contrast of volumetric OCM. This hybrid adaptive optics (hyAO) approach splits the image formation process into a combination of hardware and computation components. For sparse sample imaging, we generated astigmatism using hardware adaptive optics (HAO) to achieve a more equalized photon distribution across depth, and removed the applied aberration (and defocus) via computational adaptive optics (CAO). We applied this hyAO method to perform 3D time-lapse imaging of in vitro fibroblast cell dynamics over a 1mm×1mm×1mm field-of-view with 2μm isotropic spatial resolution and 3-minute temporal resolution. The hyAO approach is not only beneficial for high-throughput volumetric imaging, but is also capable of suppressing MS/speckle. For scattering sample imaging, HAO was used to illuminate the sample volume with diverse aberrated point spread functions to decorrelate the MS/speckle fields, and CAO was applied to computationally mitigate the resolution penalty of these intentionally induced aberrations. By imaging with this aberration-diverse OCT using 12 volumetric reconstructions, we achieved a 10 dB enhancement in signal-to-background ratio at a USAF target plane beneath a scattering layer (7.2 scattering mean-free-path), and a 3× speckle contrast reduction within the scattering layer.

Background: Multiphoton microscopy (MPM) has enabled in vivo time-lapse imaging of the heart that shows motion of cells within the tissue with micrometer resolution. We developed automated analysis techniques to quantify cellular motion from in vivo cardiac MPM images throughout the cardiac cycle. Methods: Intravital cardiac MPM of the beating mouse heart was performed on 26 week-old, C57Bl6 mice (n=6). Image volumes (100 μm deep) were acquired at 30 frames per second while recording the electrocardiogram and respiratory pressure. An image volume was reconstructed by assembling lines acquired nearest to a specified point in the cardio-respiratory phase space (Fig. a). Motion was calculated as the three-dimensional transformation required to register the reconstructed images to the image at the most stable cardiac phase. Results: Volumes were reconstructed in 50 intervals across the cardiac phase that show vasculature (intravenous Texas-red dextran, red) and cardiomyocytes (rhodamine 6G, cyan) moving across the field of view (Fig. b). Automated analysis indicated a maximum displacement occurring at 16 % (anterior-posterior), 36 % (base-apex) and 38 % (epi-endocardial) of the cardiac cycle defined by R-wave. Comparison by manual tracking of features across the cardiac cycle at a subset of phases (10 cardiac phases at peak exhalation) validated the automated measurement (Fig. c). Automated motion tracking shows superior performance in the spatial resolution and speed of analysis. Conclusions: We have shown a novel, fast, and accurate technique for characterizing cardiac motion from in vivo cardiac MPM to study the performance of the contractile cells in health and disease.

In vivo multiphoton microscopy of the beating mouse heart generates cell-resolved, volumetric images parameterized by cardiorespiratory phase-space. We compare displacement and deformation profiles throughout the cardiac cycle before and after injury.

Background: The study of functional cardiomyocyte adaptation and inflammatory cell behavior at the micro-scale in vivo has been challenging due to limited imaging tools. We recently developed intravital multiphoton microscopy (MPM) methods that enable visualization and quantification of cardiac dynamics at a cell-scale throughout the cardiac cycle. We aimed to determine the dynamic cellular changes that occur due to high fat diet (HFD) induced hypertrophy using intravital cardiac MPM. Methods: ApoE -/- C57Bl6 mice started a HFD at 6 weeks of age (ApoE -/- -HFD, n=11), while age-matched wild-type mice (WT-ND, n=10) were fed a normal chow diet. At 26-weeks, mice were assessed by cardiac echocardiography and intravital MPM in the intact beating heart. Intravenous injections of rhodamine-6G (R6g) labeled cardiomyocytes and leukocytes, and Texas-Red dextran labeled vasculature. 3D volumes were reconstructed throughout the cardiac cycle to quantify cell motion using automated algorithms. Results: ApoE -/- -HFD hearts underwent hypertrophy compared to WT-ND with increased heart weight-to-tibial length ratio (10±0.8 vs 13±1.1) and left ventricle wall thickness (1.07±0.03 mm vs 1.13±0.06 mm, respectively, p<0.05 for both) while ejection fraction remained similar (66±3 % vs 59±3 %). In vivo MPM demonstrated that cells move a greater total distance in each cardiac cycle in ApoE -/- -HFD vs WT-ND. Maximum displacement in the apex-base and anterior-posterior directions increased by 46 % in ApoE -/- -HFD compared to WT-ND (30 μm vs 14 μm). R6g+ leukocytes were visible moving in capillaries. The incidence of patrolling behavior (defined as slowing moving cells, visible for longer than one heart beat) increased in capillaries of ApoE -/- -HFD compared to WT-ND (3.4±0.5/min vs 0.12±0.1/min, p<0.01). Conclusion: These results suggest that hypertrophied cardiomyocytes increase myocardial displacement, and increased leukocyte patrolling behavior is associated with HFD induced cardiac hypertrophy. Intravital cardiac MPM provides a novel perspective to study HFD induced cardiac hypertrophy by capturing the simultaneous contributions of inflammatory cells and myocyte function in the beating heart.

Multiple scattering is a major barrier that limits the optical imaging depth in scattering media. In order to alleviate this effect, we demonstrate aberration-diverse optical coherence tomography (AD-OCT), which exploits the phase correlation between the deterministic signals from single-scattered photons to suppress the random background caused by multiple scattering and speckle. AD-OCT illuminates the sample volume with diverse aberrated point spread functions, and computationally removes these intentionally applied aberrations. After accumulating 12 astigmatism-diverse OCT volumes, we show a 10 dB enhancement in signal-to-background ratio via a coherent average of reconstructed signals from a USAF target located 7.2 scattering mean free paths below a thick scattering layer, and a 3× speckle contrast reduction from an incoherent average of reconstructed signals inside the scattering layer. This AD-OCT method, when implemented using astigmatic illumination, is a promising approach for ultra-deep volumetric optical coherence microscopy.

Using hardware adaptive optics to introduce optical aberrations, and computational adaptive optics to compensate their resolution penalty, we perform coherent averaging of reconstructed aberration-diverse OCT datasets to suppress the effects of multiple scattering.

We present the first system penalty measurements for all-optical wavelength conversion in an integrated ring resonator. We achieve wavelength conversion over a range of 27.7nm in the C-band at 2.5 Gb/s by exploiting four wave mixing in a CMOS compatible, high index glass ring resonator at ~22 dBm average pump power, obtaining < 0.3 dB system penalty.

We demonstrate sub-picosecond wavelength conversion in the C-band via four wave mixing in a 45cm long high index doped silica spiral waveguide. We achieve an on/off conversion efficiency (signal to idler) of +16.5dB as well as a parametric gain of +15dB for a peak pump power of 38W over a wavelength range of 100nm. Furthermore, we demonstrated a minimum gain of +5dB over a wavelength range as large as 200nm.

We stabilize a frequency comb generated by supercontinuum from a Si3N4 microchip and compare it to that generated in silica photonic crystal fibers. For high effective nonlinearities, spontaneous Raman scattering in silica can significantly degrade the supercontinuum coherence.

Silicon nitride (Si3N4) waveguides represent a novel photonic platform that is ideally suited for energy efficient and ultrabroadband nonlinear interactions from the visible to the mid-infrared. Chip-based supercontinuum generation in Si3N4 offers a path towards a fully-integrated and highly compact comb source for sensing and time-and-frequency metrology applications. We demonstrate the first successful frequency comb offset stabilization that utilizes a Si3N4 waveguide for octave-spanning supercontinuum generation and achieve the lowest integrated residual phase noise of any diode-pumped gigahertz laser comb to date. In addition, we perform a direct comparison to a standard silica photonic crystal fiber (PCF) using the same ultrafast solid-state laser oscillator operating at 1 µm. We identify the minimal role of Raman scattering in Si3N4 as a key benefit that allows to overcome the fundamental limitations of silica fibers set by Raman-induced self-frequency shift.

We show that self-organization and synchronization underlie Kerr-cavity-soliton formation in parametric frequency combs. By reducing the Lugiato-Lefever equation to a set of phase equations, we find that self-organization arises from a two-stage process via pump-degenerate and pump-nondegenerate four-wave mixing. The reduced phase equations are akin to the Kuramoto model of coupled oscillators and intuitively explain the origin of the pump phase offset, predict antisymmetrization of the intracavity field before phase synchronization, and clarify the role of chaos in Kerr-cavity-soliton formation in parametric combs.