Myocardial blood flow deficits in heart failure with preserved ejection fraction (HFpEF) patients and related animal models have been recognized for decades, but the underlying mechanisms and resulting consequences for HFpEF pathogenesis remain poorly understood. Using intravital cardiac microcopy in a two-hit mouse model of HFpEF, we identified an increased number of neutrophils in capillaries slow moving orstalled, blocking blood flow, as compared to control mice. Administration of antibodies against the neutrophil marker Ly6G reduced the number of arrested neutrophils in myocardial capillaries, leading to both a reduction in myocardial tissue hypoxia and improvement in diastolic function and exercise tolerance. This study identifies a previously uncharacterized cellular mechanism that explains myocardial blood flow deficits in mouse models of HFpEF, and demonstrates that improving myocardial blood flow improves heart function. Restoring myocardial perfusion by preventing neutrophil arrest in coronary capillaries may provide a strategy for improving heart function in HFpEF patients. ### Competing Interest Statement The authors have declared no competing interest.

Background: Chronic inflammation and myocardial hypoperfusion are implicated in the pathogenesis of heart failure with preserved ejection fraction (HFpEF), but the cellular and molecular mechanisms are unknown. Intravital cardiac multiphoton microscopy (MPM) enables direct visualization of blood flow and inflammatory cells within the capillary bed of the actively beating heart. We hypothesize that transient arrest of neutrophils in myocardial capillaries leads to perfusion deficits, resulting in hypoxia and driving disease progression. Methods: Male and female 8-12-week-old C57BL/6 mice received a high fat diet and L-NAME in drinking water (HFpEF) or standard diet and water (Chow) for 15 weeks. Intravital imaging of the actively beating heart was performed in mechanically ventilated mice to visualize neutrophils and capillary perfusion. An antibody against the neutrophil surface marker Ly6G (αLy6G) was administered intraperitoneally to test the effect of neutrophil depletion. Results: In vivo MPM revealed increases in arrested neutrophils in myocardial capillaries in HFpEF mice compared to Chows. Myocardial hypoxia, assessed by pimonidazole staining, and diastolic function, assessed as mitral e/e’ using echocardiography, were rescued with both extended neutrophil depletion (2mg/kg every 3d for 4 wk) and acutely (24 hrs after a single dose of 4mg/kg). To further test the immediate effects of αLy6G we implemented multi-exposure laser speckle imaging for perfusion and spectral imaging for tissue and blood oxygenation with electrocardiograph-driven reconstruction. Conclusion: Arrested neutrophils within capillaries appear to compromise myocardial perfusion in HFpEF. Neutrophil depletion to restore blood flow improved myocardial oxygenation and diastolic function rapidly. Effects were sustained with prolonged treatment. Capillary stall perfusion deficits may be a future therapeutic target to slow disease progression and offer rapid symptomatic relief.

Understanding the response to cardiac injury across multiple cell types and timescales has relied on techniques that reduce the complexity of the in vivo environment to provide a step‐by‐step picture of heart function. These models do not recapitulate the temporal and spatial complexities of the true in vivo environment. We have developed a suite of in vivo imaging methods that enable the study of cardiac injury, and here demonstrate intravital cardiac multiphoton microscopy (MPM) to assess cardiomyocyte function and the dynamics of heart and inflammatory cell types in response to cardiac injury and repair.

We have used the novel imaging technique of intravital cardiac MPM to discover a new behavior of neutrophils within myocardial capillaries during HFpEF. We demonstrate that arrested capillary neutrophils impair myocardial perfusion to promote HFpEF. Targeting arrested neutrophil behavior offers potential therapeutic benefits.

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.

Tadalafil 40 mg once daily is approved for adult patients with pulmonary arterial hypertension (PAH). To investigate and potentially fulfill an unmet need in pediatric patients with PAH, pharmacokinetic (PK) data were explored in a pediatric phase Ib/II study and pooled with prior phase III (pulmonary arterial hypertension and response to tadalafil [PHIRST-1]) adult data to develop the first population PK model for tadalafil in pediatric patients with PAH. H6D-MC-LVIG (NCT01484431) was an open-label, multicenter, multiple ascending dose study in pediatric patients with PAH, while PHIRST-1 was a phase III, multicenter, randomized, double-blind, placebo-controlled, parallel design study in adults with PAH who received one of five treatments (tadalafil 2.5, 10, 20, or 40 mg, or placebo orally, once daily). PK data from the studies were pooled to develop a pediatric population PK model for tadalafil that characterized relationships among dose, exposure, and the effects of covariates with an aim to develop a population PK model that could simulate concentration–time profiles and assess exposure-matched dosing strategies in a pediatric PAH population. In line with the observed data, modeling and simulation demonstrated that the doses studied in the pediatric population produced area under the concentration–time curves (AUCs) within the range of those associated with improved exercise ability in adults with PAH. The analyses included 1430 observations from 305 adult patients (PHIRST-1: 69 males and 236 females, 1102 observations) and 19 pediatric patients (LVIG: 6 males and 13 females, 328 observations) who received tadalafil once daily at different dose levels. The best-fit base model retained an effect of weight on apparent volume of distribution (V/F), fixed to the allometric scaling value of 1, and did not include an effect of weight on apparent clearance (CL/F). Other covariate effects were that bosentan increased CL/F, V/F decreased with decreasing body weight, and bioavailability (F) decreased with increasing dose and decreasing age. The PK model reliably predicted the observed concentrations and overall variability evident from the overlap of the individual observed concentrations with the distributions of simulated concentrations. A one-compartment model parameterized in terms of F, absorption rate constant, CL/F, and V/F described the data well. The model demonstrated that plasma tadalafil concentrations in pediatric patients aged 2 to < 18 years were similar to those in adults at similar doses, and confirmed that dosing of 40 mg once daily in pediatric patients with a bodyweight ≥ 40 kg, and a dose of 20 mg once daily in patients with a body weight < 40 kg and aged ≥ 2 years are suitable for phase III evaluation. LVIG: ClinicalTrials.gov identifier: NCT01484431 (2 December 2011). PHIRST-1: ClinicalTrials.gov identifier: NCT00125918 (2 August 2005).

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.

Abstract Introduction One of the major neuropathological features of Alzheimer's disease (AD) is the accumulation of amyloid‐β (Aβ) protein in the brain. Evidence suggests that the low‐density lipoprotein receptor‐associated protein (RAP) binds strongly to Aβ and enhances its cellular uptake and that decreased RAP expression correlates with increased Aβ production in animal models of AD. Methods The current study examined whether RAP levels change in AD human brain tissue and whether they are related to the amount of AD pathology. RAP and NeuN levels were determined by Western blot, while low‐density lipoprotein receptor‐related protein 1 (LRP1), tau and Aβ levels were determined by ELISA in the temporal cortex of 17 AD and 16 control cases. Results An increase in total Aβ and insoluble and soluble tau protein was observed in AD brain tissue. In contrast, RAP levels were significantly decreased in AD brain tissue compared to controls. Correlation analysis revealed that levels of RAP correlated with both total Aβ and soluble and insoluble tau levels. Neither LRP1 nor NeuN levels were significantly altered in AD brain tissue homogenates and did not correlate with Aβ or tau protein levels. Conclusion Reduction in RAP may contribute to the accumulation and aggregation of Aβ in the AD brain.

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.

The β-site APP Cleaving enzyme 1 (BACE1) is a membrane-associated aspartyl protease which mediates the production of amyloid-β (Aβ), a major component of amyloid plaques in the Alzheimer’s disease brain. We have synthesised and characterised a series of peptidomimetic analogues of BACE substrates that incorporate two distinct stabilising structures. To demonstrate the potential activity of these compounds, a variety of assaying strategies were used to investigate cleavage susceptibility and inhibition potency under competitive and non-competitive conditions. β-Amino acids and scissile site N-methylation were incorporated into peptide substrate templates as transition state isostere (TSI) substitutes by positional scanning to generate series of non-TSI β-peptidomimetics. The amino acid sequences flanking the β-cleavage site within APP carrying the Swedish double mutation (APPSW), Neuregulin, the synthetic hydroxyethylene-based TSI peptide inhibitor OM99-2, and the high affinity peptide sequence SEISYEVEFR, served as the four substrate templates from which over 60 peptides were designed and synthesised by solid phase peptide synthesis. A quenched fluorescent substrate BACE1 assay in conjunction with liquid chromatography–mass spectrometry (LC-MS) analysis was established to investigate cleavage susceptibility and inhibition potency under competitive and non-competitive conditions. It was determined that β-amino acids substituted at the P1 scissile site position within known peptide substrates were resistant to proteolysis, and particular substitutions induced a concentration-dependent stimulation of BACE1, indicating a possible modulatory role of native BACE1 substrates.

Abstract. The compromise between lateral resolution and usable imaging depth range is a bottleneck for optical coherence tomography (OCT). Existing solutions for optical coherence microscopy (OCM) suffer from either large data size and long acquisition time or a nonideal point spread function. We present volumetric OCM of mouse brain ex vivo with a large depth coverage by leveraging computational adaptive optics (CAO) to significantly reduce the number of OCM volumes that need to be acquired with a Gaussian beam focused at different depths. We demonstrate volumetric reconstruction of ex-vivo mouse brain with lateral resolution of 2.2  μm, axial resolution of 4.7  μm, and depth range of ∼1.2  mm optical path length, using only 11 OCT data volumes acquired on a spectral-domain OCM system. Compared to focus scanning with step size equal to the Rayleigh length of the beam, this is a factor of 4 fewer datasets required for volumetric imaging. Coregistered two-photon microscopy confirmed that CAO-OCM reconstructions can visualize various tissue microstructures in the brain. Our results also highlight the limitations of CAO in highly scattering media, particularly when attempting to reconstruct far from the focal plane or when imaging deep within the sample.

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.

AimsTo evaluate the pharmacokinetics and safety of once‐daily (QD) tadalafil in paediatric patients with pulmonary arterial hypertension (PAH) to establish an appropriate dose range for further research.MethodsThis was an open‐label, multicentre, international, multiple‐ascending‐dose study. Patients aged ≥2 years were enrolled into 1 of 3 cohorts based on body weight: heavy‐weight (≥40 kg), middle‐weight (25 to <40 kg), and light‐weight (<25 kg). Each patient received tadalafil QD for 10 weeks: 5 weeks at a low dose, then 5 weeks at a high dose. The doses for each cohort were intended to produce plasma tadalafil concentrations within the range produced by 5–10 mg (for the low dose) or 20–40 mg (for the high dose) of tadalafil in adults with PAH. Area under the plasma concentration–time curve during 1 dosing interval (AUCτ), maximum concentration, and apparent clearance were assessed throughout the trial, as were safety and tolerability.ResultsThe study enrolled 19 patients aged 2–17 years, weighing 9.9–76.0 kg. Tadalafil's median (range) steady‐state AUCτ at the high dose was 7243 (3131–13 088) ng•h/mL across all patients. Concentrations were higher in no bosentan‐treated patients than in bosentan‐treated patients, but both populations were within the range of respective adult patients taking 20–40 mg QD. Tadalafil had an acceptable safety profile consistent with the known safety profile of tadalafil in adults.ConclusionsTadalafil 40 mg QD for patients ≥40 kg, and 20 mg QD for patients <40 kg and aged ≥2 years, are suitable for further research in paediatric patients with PAH.

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.

Intravital microscopy is a powerful technique to observe dynamic processes with single-cell resolution in live animals. No intravital window has been developed for imaging the colon due to its anatomic location and motility, although the colon is a key organ where the majority of microbiota reside and common diseases such as inflammatory bowel disease, functional gastrointestinal disorders, and colon cancer occur. Here we describe an intravital murine colonic window with a stabilizing ferromagnetic scaffold for chronic imaging, minimizing motion artifacts while maximizing long-term survival by preventing colonic obstruction. Using this setup, we image fluorescently-labeled stem cells, bacteria, and immune cells in live animal colons. Furthermore, we image nerve activity via calcium imaging in real time to demonstrate that electrical sacral nerve stimulation can activate colonic enteric neurons. The simple implantable apparatus enables visualization of live processes in the colon, which will open the window to a broad range of studies.

The function of the β-A4 amyloid protein precursor (APP) of Alzheimer's disease (AD) remains unclear. APP has a number of putative roles in neuronal differentiation, survival, synaptogenesis and cell adhesion. In this study, we examined the development of axons, dendrites and synapses in cultures of hippocampus neutrons derived from APP knockout (KO) mice. We report that loss of APP function reduces the branching of cultured hippocampal neurons, resulting in reduced synapse formation. Using a compartmentalised culture approach, we found reduced axonal outgrowth in cultured hippocampal neurons and we also identified abnormal growth characteristics of isolated hippocampal neuron axons. Although APP has previously been suggested to play an important role in promoting cell adhesion, we surprisingly found that APPKO hippocampal neurons adhered more strongly to a poly-L-lysine substrate and their neurites displayed an increased density of focal adhesion puncta. The findings suggest that the function of APP has an important role in both dendritic and axonal growth and that endogenous APP may regulate substrate adhesion of hippocampal neurons. The results may explain neuronal and synaptic morphological abnormalities in APPKO mice and the presence of abnormal APP expression in dystrophic neurites around amyloid deposits in AD.

Aim: Evaluate steady-state pharmacokinetics and potential interactions between select statins and evacetrapib. Patients & methods: This open-label, two-part study included 62 healthy native Chinese subjects. Part 1 evaluated pharmacokinetics and pharmacodynamics of evacetrapib after 1 or 14 once-daily doses. Part 2 evaluated pharmacokinetics and pharmacodynamics of simvastatin, atorvastatin and evacetrapib administered alone, and of statin + evacetrapib coadministered. Results: Evacetrapib estimated accumulation ratio following once-daily dosing was 2.7. Simvastatin or atorvastatin coadministration reduced evacetrapib AUC0–24 by 12% (90% CI: -0.1 to -22%) or 10% (90% CI: -23–5%), respectively. Evacetrapib coadministration increased simvastatin or atorvastatin AUC0–24 by 123% (90% CI: 91–159%) or 16% (90% CI: 6–27%), respectively. Evacetrapib administered alone and with a statin increased high-density lipoprotein cholesterol, decreased low-density lipoprotein cholesterol, and was well tolerated. Conclusion: The significant increase in simvastatin exposure after evacetrapib coadministration was unexpected compared with previous evacetrapib and simvastatin interaction studies.