Mapping by VESGEN of Blood Vessels in the Retinas of ISS Crew Members and Bed Rest Subjects for Increased Understanding of VIIP

Research by NASA has established that significant risks for visual impairment in association with increased intracranial pressure (VIIP) are incurred by microgravity spaceflight, especially long-duration missions. Impairments include decreased near visual acuity, posterior globe flattening, choroidal folds, optic disc edema, and cotton wool spots. Much remains to be learned about the etiology of VIIP before effective countermeasures can be developed. Contributions of retinal vascular remodeling to the etiology of VIIP have not yet been investigated, primarily due to the current lack of ophthalmic tools for precisely measuring progressive pathophysiological remodeling of the retinal microvasculature. Although ophthalmic science and clinical practice are now highly sophisticated at detecting indirect, secondary signs of vascular remodeling such as cotton wool spots that arise during the progression of retinal vascular diseases, methods for quantifying direct, primary vascular changes are not yet established. To help develop insightful analysis of retinal vascular remodeling for aerospace medicine, we will map and quantify by our innovative VESsel GENeration Analysis (VESGEN) software the remodeling status of retinal blood vessels in crew members before and after ISS missions, and in healthy human subjects before and after head-down tilt bed rest. For this proof-of-concept study, we hypothesize that pathophysiological remodeling of retinal blood vessels occurs in coordination with microgravity-induced fluid shifts prior to development of visual impairments. VESGEN analysis in previous research supported by the US National Institutes of Health identified surprising new opportunities to regenerate retinal vessels during early-stage progression of the visually impairing, potentially blinding disease, diabetic retinopathy

Hindlimb Suspension (HLS) in Rodents for the Study of Intracranial Pressure, Molecular and Histologic Changes in the Eye, and CSF Production Regulation and Resorption: A Status Report of Two Studies

This status report corresponds to two studies tied to an animal experiment being executed at the University of California Davis (Charles Fuller's laboratory). The animal protocol uses the well-documented rat hindlimb suspension (HLS) model, to examine the relationship between cephalic fluid shifts and the regulation of intracranial (ICP) and intraocular (IOP) pressures as well as visual system structure and function. Long Evans rats are subjected to HLS durations of 7, 14, 28 and 90 days. Subgroups of the 90-day animals are studied for recovery periods of 7, 14, 28 or 90 days. All HLS subjects have age-matched cage controls. Various animal cohorts are planned for this study: young males, young females and old males. In addition to the live measures (ICP by telemetry, IOP and retinal parameters by optical coherence tomography) which are shared with the Fuller study, the specific outcomes for this study include: -Gene expression analysis of the retina -Histologic analysis - Analysis of the microvasculature of retina flat mounts by NASA's VESsel GENeration Analysis (VESGEN) Software. To date, the young male and female cohorts are being completed. Due to the need to keep technical variation to a minimum, the histologic and genomic analyses have been delayed until all samples from each cohort are available and can be processed in a single batch per cohort. The samples received so far correspond to young males sacrificed at 7,14, 28 and 90 days of HLS and at 90 days of recovery; and from young females sacrificed at 7, 14 and 28 of HLS. A complementary study titled: "A gene expression and histologic approach to the study of cerebrospinal fluid (CSF) production and outflow in hindlimb suspended rats" seeks to study the molecular components of CSF production and outflow modulation as a result of HLS, bringing a molecular and histologic approach to investigate genome wide expression changes in the arachnoid villi and choroid plexus of HLS rats compared to rats in normal posture

VESGEN Mapping of Bioactive Protection against Intestinal Inflammation: Application to Human Spaceflight and ISS Experiments

Challenges to successful space exploration and colonization include adverse physiological reactions to micro gravity and space radiation factors. Constant remodeling of the microvasculature is critical for tissue preservation, wound healing, and recovery after ischemia. Regulation of the vascular system in the intestine is particularly important to enable nutrient absorption while maintaining barrier function and mucosal defense against micro biota. Although tremendous progress has been made in understanding the molecular circuits regulating neovascularization, our knowledge of the adaptations of the vascular system to environmental challenges in the intestine remains incomplete. This is in part because of the lack of methods to observe and quantify the complex processes associated with vascular responses in vivo. Developed by GRC as a mature beta version, pre-release research software, VESsel GENeration Analysis (VESGEN) maps and quantifies the fractal-based complexity of vascular branching for novel insights into the cytokine, transgenic and therapeutic regulation of angiogenesis, lymphangiogenesis and microvascular remodeling. Here we demonstrate that VESGEN can be used to characterize the dynamic vascular responses to acute intestinal inflammation and mucosal recovery from in vivo confocal microscopic 3D image series. We induced transient intestinal inflammation in mice by DSS treatment and investigated whether the ability of the pro biotic yeast Saccharomyces boulardii (Sb) to protect against intestinal inflammation was due to regulation of vascular remodeling. A primary characteristic of inflammation is excessive neovascularization (angiogenesis) resulting in fragile vessels prone to bleeding. Morphological parameters for triplicate specimens revealed that Sb treatment greatly reduced the inflammatory response of vascular networks by an average of 78%. This resulted from Sb inhibition of vascular endothelial growth factor receptor signaling, a major angiogenesis signaling pathway. It needs to be determined whether pro biotic yeast represents a promising approach to GI protection in space. GRC performed only the VESGEN post-testing analysis

Differences in Pre and Post Vascular Patterning of Retinas from ISS Crew Members and HDT Subjects by VESGEN Analysis

Accelerated research by NASA [1] has investigated the significant risks for visual and ocular impairments Spaceflight Associated Neuro-Ocular Syndrome /Visual Impairment/Intracranial Pressure (SANS/VIIP) incurred by microgravity spaceflight, especially long-duration missions. Our study investigates the role of blood vessels in the incidence and etiology of SANS/VIIP within the retinas of Astronaut crewmembers pre-and post-flight to the International Space Station (ISS) by NASA's VESsel GENeration Analysis (VESGEN). The response of retinal vessels in crewmembers to microgravity was compared to that of retinal vessels to Head-Down Tilt (HDT) in subjects undergoing 70-Day Bed Rest. The study tests the proposed hypothesis that cephalad fluid shifts missions, resulting in ocular and visual impairments, are necessarily mediated in part by retinal blood vessels, and are therefore accompanied by significant remodeling of retinal vasculature.Vascular patterns in the retinas of crew members and HDTBR subjects extracted from 30 infrared (IR) Heidelberg Spectralis images collected pre/postflight and pre/post HDTBR, respectively, were analyzed by VESGEN (patent pending). a mature, automated software developed as a research discovery tool for progressive vascular diseases in the retina and other tissues [2]. The weighted, multi-parametric VESGEN analysis generates maps of branching arterial and venous trees and quantification by parameters such as the fractal dimension (Df, a modern measure of vascular space-filling capacity), vessel diameters, and densities of vessel length and number classified into specific branching generations by vascular physiological branching rules [2,3]. The retrospective study approved by NASAs Institutional Review Board included six HDT subjects (NASA Flight Analogs Research Unit [FARU] Campaign 11; for example, [4]) and eight ISS crewmembers monitored by routine occupational surveillance who provided their study consents to NASAs Lifetime Surveillance of Astronaut Health (LSAH). For the initial blinded VESGEN phase, ophthalmic retinal images were masked as to subject identity and pre- and post-status. In the second unblinded phase, VESGEN results were analyzed according to the pre- and post-status of left and right retinas matched to each subject. To complete our study, vascular results will be subjected to NASA biostatistical analysis and correlated with other ophthalmic and medical findings. Preliminary results for changes in the pre- to post-status of vascular patterning in the retinas of crewmembers and HDT subjects are strikingly opposite. By Df and other vascular branching measures, the space-filling capacity of arterial and venous trees decreased in a substantial subset of crewmembers (11/16 retinas). In contrast, vascular densities increased in a substantial subset of HDT subjects by the same parameters (6/10 retinas, currently excluding one anomalous subject). To conclude the study, biostatistical and medical analyses will be of critical importance for investigating the validity of these vascular findings. Vascular densities appeared to decrease in the retinas of crewmembers following ISS Missions, and increase in subjects after HDT. The vascular increases and decreases most likely derive primarily from limits of resolution to the ophthalmic imaging that does not capture the smallest vessels, rather than from vessel growth or atrophy. Differences in arterial and venous response to cephalad fluid shifts induced by ISS and HDT may have resulted from a long-duration conditioning phenomenon (for example, 6-month ISS missions compared to 70-day HDT), or the presence of gravity in HDT compared to microgravity onboard the ISS. To conclude our study, the biostatistical and medical analyses will be of critical importance for investigating the validity and significance of the VESGEN findings

Microvascular Branching as a Determinant of Blood Flow by Intravital Particle Imaging Velocimetry

The effects of microvascular branching on blood flow were investigated in vivo by microscopic particle imaging velocimetry (micro-PIV). We use micro-PIV to measure blood flow by tracking red blood cells (RBC) as the moving particles. Velocity flow fields, including flow pulsatility, were analyzed for the first four branching orders of capillaries, postcapillary venules and small veins of the microvascular network within the developing avian yolksac at embryonic day 5 (E5). Increasing volumetric flowrates were obtained from parabolic laminar flow profiles as a function of increasing vessel diameter and branching order. Maximum flow velocities increased approximately twenty-fold as the function of increasing vessel diameter and branching order compared to flow velocities of 100 - 150 micron/sec in the capillaries. Results from our study will be useful for the increased understanding of blood flow within anastomotic, heterogeneous microvascular networks

Mapping by VESGEN of Blood Vessels in the Retinas of Astronauts Pre- and Post-Flight to the ISS

Research by NASA [1] established that significant risks for visual and ocular impairments associated with increased intracranial pressure (VIIP) are incurred by microgravity spaceflight, especially long-duration missions. It is well established in physiology and pathology that a fundamental role of the microvasculature is to mediate fluid transfers and remodel actively in response to environmental, immune and other stresses. We therefore hypothesize that remodeling of retinal blood vessels necessarily occurs during accommodation of microgravity-induced fluid shifts prior to subsequent development of visual and ocular impairments. Potential contributions of retinal vascular remodeling to VIIP etiology are therefore being investigated by NASA's innovative VESsel GENeration Analysis (VESGEN) software for two studies: (1) U.S. crew members before and after ISS missions, and (2) head-down tilt in human subjects before and after 70 days of bed rest. We anticipate that results of the two studies will be complete by the Investigators Workshop (January 22, 2017). METHODS: For the 2013 NASA NRA award, we are concluding the analysis of 30 degree infrared (IR) Heidelberg Spectralis images of retinal blood vessels by VESGEN (patents pending), a mature, automated software developed as a translational and basic vascular research discovery tool, particularly for retinal vascular disease. Subjects of our retrospective study include eight ISS crew members monitored for routine occupational surveillance pre- and post-flight, who provided their study consents to NASAs Lifetime Surveillance of Astronaut Health (LSAH) in coordination with approval of the VESGEN retrospective study protocol by NASAs Institutional Review Board (IRB). The ophthalmic retinal images (average image resolution, approximately 5.6 microns per pixel) are blinded as to pre and post ISS status until the second portion of our study, when VESGEN results will be correlated with other ophthalmic and medical findings for the crew members. Due to image resolution challenges, a novel Matlab tool was developed for aligning pre and post images, and comparing (querying) the two images for differences in the morphology of small vessels. RESULTS: During the past year, LSAH approved the release of all astronaut retinal images to our study for VESGEN analysis. Substantial progress on the initial blinded portion of the study is in place. We anticipate that VESGEN analysis of the 32 Spectralis IR retinal images will be complete for presentation at the 2017 IWS meeting. CONCLUSIONS: Modified retinal vascular patterning may offer early-stage predictions of ocular changes resulting in decreased visual acuity for the VIIP syndrome. Novel insights provided by VESGEN into progressively pathological and blinding vascular remodeling in the human retina currently help to guide other NIH- and NASA-supported therapeutic studies of retinal disease and modeling of the VIIP risk. Results of our vascular investigation of the retinas of astronauts pre- and post-flight may help advance the understanding of both healthy and pathological adaptations to fluid shifts in microgravity associated with the VIIP syndrome. Preliminary results indicate that imaging of higher resolution, such as the new OCT angiography (OCT-A) technology, will be required to determine conclusively the role of the smaller retinal and choroidal vessels in VIIP etiology

Rapid Analysis of Vessel Elements (RAVE): A Tool for Studying Physiologic, Pathologic and Tumor Angiogenesis

Quantification of microvascular network structure is important in a myriad of emerging research fields including microvessel remodeling in response to ischemia and drug therapy, tumor angiogenesis, and retinopathy. To mitigate analyst-specific variation in measurements and to ensure that measurements represent actual changes in vessel network structure and morphology, a reliable and automatic tool for quantifying microvascular network architecture is needed. Moreover, an analysis tool capable of acquiring and processing large data sets will facilitate advanced computational analysis and simulation of microvascular growth and remodeling processes and enable more high throughput discovery. To this end, we have produced an automatic and rapid vessel detection and quantification system using a MATLAB graphical user interface (GUI) that vastly reduces time spent on analysis and greatly increases repeatability. Analysis yields numerical measures of vessel volume fraction, vessel length density, fractal dimension (a measure of tortuosity), and radii of murine vascular networks. Because our GUI is open sourced to all, it can be easily modified to measure parameters such as percent coverage of non-endothelial cells, number of loops in a vascular bed, amount of perfusion and two-dimensional branch angle. Importantly, the GUI is compatible with standard fluorescent staining and imaging protocols, but also has utility analyzing brightfield vascular images, obtained, for example, in dorsal skinfold chambers. A manually measured image can be typically completed in 20 minutes to 1 hour. In stark comparison, using our GUI, image analysis time is reduced to around 1 minute. This drastic reduction in analysis time coupled with increased repeatability makes this tool valuable for all vessel research especially those requiring rapid and reproducible results, such as anti-angiogenic drug screening

Position resolution and particle identification with the ATLAS EM calorimeter

In the years between 2000 and 2002 several pre-series and series modules of the ATLAS EM barrel and end-cap calorimeter were exposed to electron, photon and pion beams. The performance of the calorimeter with respect to its finely segmented first sampling has been studied. The polar angle resolution has been found to be in the range 50-60 mrad/sqrt(E (GeV)). The neutral pion rejection has been measured to be about 3.5 for 90% photon selection efficiency at pT=50 GeV/c. Electron-pion separation studies have indicated that a pion fake rate of (0.07-0.5)% can be achieved while maintaining 90% electron identification efficiency for energies up to 40 GeV.Comment: 32 pages, 22 figures, to be published in NIM

Energy Linearity and Resolution of the ATLAS Electromagnetic Barrel Calorimeter in an Electron Test-Beam

A module of the ATLAS electromagnetic barrel liquid argon calorimeter was exposed to the CERN electron test-beam at the H8 beam line upgraded for precision momentum measurement. The available energies of the electron beam ranged from 10 to 245 GeV. The electron beam impinged at one point corresponding to a pseudo-rapidity of eta=0.687 and an azimuthal angle of phi=0.28 in the ATLAS coordinate system. A detailed study of several effects biasing the electron energy measurement allowed an energy reconstruction procedure to be developed that ensures a good linearity and a good resolution. Use is made of detailed Monte Carlo simulations based on Geant which describe the longitudinal and transverse shower profiles as well as the energy distributions. For electron energies between 15 GeV and 180 GeV the deviation of the measured incident electron energy over the beam energy is within 0.1%. The systematic uncertainty of the measurement is about 0.1% at low energies and negligible at high energies. The energy resolution is found to be about 10% sqrt(E) for the sampling term and about 0.2% for the local constant term

A new measurement of direct CP violation in two pion decays of the neutral kaon

The NA48 experiment at CERN has performed a new measurement of direct CP violation, based on data taken in 1997 by simultaneously collecting K_L and K_S decays into pi0pi0 and pi+pi-. The result for the CP violating parameter Re(epsilon'/epsilon) is (18.5 +/- 4.5(stat)} +/- 5.8 (syst))x10^{-4}.Comment: 18 pages, 6 figure