Over time, the reaction rate decreased and eventually plateaued to a rate similar to the U + H2O + O2 reaction. This behaviour was not attributed to oxygen ingress, but instead the decreasing permeability of the grout, limiting oxidising species access to the metal surface.Uranium encapsulated in grout was exposed to water vapour for extended periods of time. Through synchrotron x-ray powder diffraction and tomography measurements, uranium dioxide was determined the dominant corrosion product over a 50-week time period. The oxide growth rate initiated rapidly, with rates comparable to the U + H The container is then capped with a secondary layer of grout and a mild steel lid, with an air vent to allow gas exchange during grout curing and corrosion of metals. The uranium metal surface may be pre-corroded, exhibiting a thick oxide layer, or, if it is grouted immediately after de-canning from the Magnox cladding, it may retain a fresh metallic surface with limited oxide development. These containers are in dry storage at Sellafield for up to 30 years, where the temperature is generally regulated. Due to Sellafield’s location, the air has a high humidity and contains salt; however, the accessibility of this atmospheric water to the metals within the grout is unknown.In the UK, Intermediate Level nuclear Waste (ILW) canisters contain primarily uranium, aluminium and Magnox alloy swarf encapsulated in a high alkaline grout (pH 10–13) within a stainless steel container. This grout is commonly composed of Blast Furnace Slag (BFS) and Ordinary Portland Cement (OPC) in a 3:1 ratio with 0.4 w/c2+x will form, and with prolonged exposure to oxygen, the layer will gradually thicken to form higher oxides (x = 0.06–1) such as U3O8 and UO35. It is well established that because uranium has a comparatively large atomic size to oxygen, its lattice diffusion within the oxide is finite6. Consequently, oxygen ions are recognised as the mobile species and new oxide develops at the oxide-metal interface7. Since oxygen ions must diffuse through the existing oxide to reach the metal, the progressively thicker oxide layer becomes an effective barrier and as such, will significantly slow uranium oxidation rates in air. In comparison, uranium oxidation in water or water vapour is observed to be distinctly faster, producing an oxide of higher porosity and friability as well as a stoichiometry closer to pure UO212. The basic equations with the respective activation energies for each scenario are14:Uranium has a high affinity for oxidising species. Immediately upon exposure to air at room temperature, a thin layer of hyper stoichiometric UO15, Equation 12, quickly converting the unstable uranium metal into a stabilised material. (2) It has the potential to form uranium hydride (UH3) at the metal-oxide interface20. Finally, (3) it releases hydrogen gas that may become trapped within the grout and later react with the uranium to form uranium hydride via Equation 21.In the context of nuclear waste storage22. In addition, the volume expansion associated with both uranium oxide and hydride formation may be sufficient to cause grout fracturing and deformation, or rupturing of the container walls, posing a risk to workers and the environment during storage and transport. However, establishing a risk assessment and quantitative analysis of metal corrosion hidden within grout and stainless steel poses a significant challenge.Uranium hydride is particularly undesirable since evidence suggests it is a pyrophoric powder27 and few studies have investigated the oxidation behaviour of uranium in grout. The most notable are a Serco report28 summarising a number of grout-uranium studies, most of which are unavailable in the open literature, and Wellman29, who focuses on the interaction of uranium with grout matrices, specifically determining the solubility limiting phases of uranium in aqueous grout equilibrated conditions. In the Serco report Godfrey30 studied the oxidation of uranium in BFS-OPC grout and concluded that uranium-grout oxidation corroded at a rate similar to Equation. 28. This latter observation was also supported by Hayes31. In contrast, other authors have shown that slow diffusivity of oxidising species from the air through the grout limits metal oxidation33; BFS was ultimately chosen for its low permeability qualities34. However, a model created by Serco to assess the potential for gas generation within an ILW container called SMOGG (Simplified Model of Gas Generation) assumed periods of oxidising conditions within the waste container during transport and storage, suggesting that gas and water vapour diffusion occurs freely through the vented container and grout28. In conclusion, the exact chemical and physical conditions within ILW grout are debatable and hard to distinguish since hydration will alter the grout chemical composition and physical properties over time. Furthermore, little research has been performed on uranium corrosion in anoxic grout conditions despite that BFS is known to form chemically reducing conditions35.Current existing literature of uranium corrosion has predominantly been performed on unconfined, bare uranium metalin situ.The aim of our study was to examine the corrosion behaviour of as-received (pre-corroded) and nitric acid etched uranium metal encapsulated in BFS:OPC grout when exposed to water vapour over a 50 week time period. These conditions were chosen to reflect the environmental conditions found in dry interim storage, and the results provide important information such as the dominant types and rates of uranium corrosion which could be used for predictive corrosion modelling of ILW containers. Synchrotron x-ray powder diffraction (XRPD) and tomography (XRT) were used to analyse the uranium encapsulated in grout, In total, 8 samples of uranium metal (rods measuring 0.5 mm × 0.5 mm × 20 mm) were encapsulated in grout and exposed to water vapour for a specific period of time (1 to 50 weeks). All samples were examined once or twice over two sessions at the Diamond Light Source (DLS), on the I12 Joint Engineering, Environment and Processing beamline (JEEP). The samples were split into two groups, described in Table 2 was identified as the dominant corrosion product in all samples, and no UH3 was detected over the 50-week period.On each sample XRPD line scan data were averaged and are displayed in Fig. 2 was detected by XRPD, however previous SIMS analysis also indicated carbon contamination  of (3.96 × 10−7)T−0:86, where T = time (weeks)  Fig. . It is b2 and H = H2O). Usually, these materials grow around grains and volumetrically reduce the grout pore network over the first 2 months of development39. BFS was chosen particularly for its ability to reduce the grout permeability (0.3 × 10−13 m.s−1 40 in comparison to 1.0 × 10−13 m.s−1 of pure OPC41), as well as its low temperature of hydration and high fluidity which together act to reduce metal oxidation rates, fill all available spaces around the uranium and ultimately reduce gas (O2 or H2O(g)) diffusion pathways to and from the metal surface1. Consequently, this was expected to have greatly affected the uranium-grout oxidation system.Hydration and maturation of OPC and BFS typically involves the development of mineral and C-S-H phases  All uranium described here originated from Magnox Ltd. The uranium rods used for Group ‘A’  were cured in their mould for 3 days in a moist environment before transfer to their reaction cells. All corrosion environments were set up in a clean test tube at room temperature for the designated period of time. Within the test tube was 5 ml of de-ionised water and the grouted sample was placed on top of a stainless steel M6 bolt. The top of each test tube was wrapped in parafilm. Before transfer to DLS, each sample was removed from its environment and remounted in a customised, hermetically sealed, quartz glass – stainless steel cell and evacuated to 1 × 1044 was used to view and reconstruct the XRT images of each sample and Avizo® was used to produce 3D renders of the XRT data using the generate surface module for specific ranges in greyscale (X-ray intensity) representing each examined material.X-ray Powder Diffraction and X-ray Tomography were performed on each sample separately on the I12 (JEEP) beamline, Diamond Light Source. Two sets of beam time were used to examine all samples. For the first session, energies of 114.6 keV and 115.6 keV were used for XRPD and XRT respectively, however this was reduced to 113.3 keV for both techniques in the second session since this energy was further away from the uranium K absorption edge (115.6 keV) and thus produced sharper XRT results. 2D XRPD data were recorded using a flat panel Pixium RF4343  in high resolution mode (2880 × 2881 pixels). This detector has a pixel size of 148 × 148 μm and beam size of ~340 × 340 μm. The high resolution PCO pco.4000 imaging detector with its Module 4 camera was used for imaging using a monochromatic beam to obtain the best resolution, 1 pixel = 0.98 × 0.98 μm. Data Analysis WorkbeNch (DAWN) software2 calibrant . Since the uranium rod position varied within the grout for each sample, the sample to detector distance varied by 1–2 mm from the central CeO2 position, thus small shifts in the 2θ value of the XRPD peaks were expected when comparing data. DAWN software for 2D diffraction and processing tools45 were used to convert data from 2D powder diffraction patterns to 1D.On each sample, two horizontal XRPD line scans were performed at different heights across the 0.5 mm width of the metal. To identify the corrosion phases, beam calibration was required once per beam time using a CeOhttps://doi.org/10.5281/zenodo.834900, and used under the Creative Commons Attribution licence.Data underlying this article can be accessed on Zenodo at Supplementary information


Maratus robinsoni and M. chrysomelas) court females using tiny structured scales (~ 40 × 10 μm2) that reflect the full visual spectrum. Using TEM and optical modelling, we show that the spiders’ scales have 2D nanogratings on microscale 3D convex surfaces with at least twice the resolving power of a conventional 2D diffraction grating of the same period. Whereas the long optical path lengths required for light-dispersive components to resolve individual wavelengths constrain current spectrometers to bulky sizes, our nano-3D printed prototypes demonstrate that the design principle of the peacock spiders’ scales could inspire novel, miniature light-dispersive components.Colour produced by wavelength-dependent light scattering is a key component of visual communication in nature and acts particularly strongly in visual signalling by structurally-coloured animals during courtship. Two miniature peacock spiders ( Several animals and plants get their often spectacular colouration and iridescence from structural colouration. Here, Hsiung et al. show the 3-dimensional nanostructures that produce the rainbow colours on the abdomen of miniature peacock spiders. Biogenic photonic nanostructures have high translational potential4 and reveal a diverse array of structural colour production mechanisms in plants and animals, including spiders12. In particular, some Australian peacock spiders can display extremely angle-dependent full-spectrum iridescence with high purity13.Controlling light through photonic micro- and nanostructures can transform human technology, including communications, sensing, security, and computingMorpho butterflies are mostly blue15. The few organisms that exhibit a rainbow pattern, such as Bronzewing pigeons (Phaps spp.), do so using spatially segregated nanostructures along each feather, creating a gradient of colours from blue to red16. Thus, the colour of any particular point on the feather does not shift between discrete hues with change in viewing angle. Other structurally coloured feathers usually shift between only a few hues19 and do not cover all colours in the visible spectrum. In contrast, the colour of abdominal scales from males of two miniature Australian peacock spiders, Maratus robinsoni . These two species of peacock spiders (Maratus spp.) raise and wiggle their abdomens toward potential mates during courtship to display every colour across the entire visible spectrum  reflective diffraction grating structures. Two-dimensional (2D) diffraction grating-like structures are not as rare as previously thought, but are still uncommon in nature, and occur only in a handful of extant and fossil species23, including plants26. Moreover, these previously described 2D diffraction gratings are likely epiphenomena that do not function in signalling, and are not then products of natural selection for optical functions25. Rainbow-iridescence is clearly a visual courtship signal in peacock spiders , hyperspectral imaging (HSI), and imaging scatterometry. We then use analytical and finite-element optical simulation to identify the mechanism of colour production in the scales. Finally, we provide validation of the mechanism using bioinspired, actual-size physical prototypes via two-photon nanolithography. Our study identifies the mechanisms by which miniature Australian peacock spider actively display isolated wavelengths within visible spectrum during their courtship. This result is significant both because it is the first insight into this mechanism and because it could provide inspiration for the development of miniature light-dispersive components. By understanding biological design principles and emulating them through engineering, our research may allow light-dispersive components to perform under irradiances, and millimetre length scales, not possible before.We hypothesize that the unique rainbow-iridescence in M. robinsoni and M. chrysomelas have two types of visually distinct abdominal scales: rainbow-iridescent scales and velvet black scales , and a depth of 300 nm, but with different shapes/geometries: the first configuration is a conventional 2D grating  and plot the simulated reflectance spectra of the three designed structures against their scattering/viewing angles Fig. . The foiM. robinsoni and M. chrysomelas diffraction gratings , which is an integer for a horizontal period of dh, and θi, θs are the incident and scattering (diffraction) angles, respectively. The reverse diffraction order can be explained by implementing the vertical grating equation and considering an exact vertical orientation of the surface grating to the surface normal29:Here, θi =0), the diffraction wavelength peak for a specific order varies proportionally to the cosine of the diffraction angle, which explains the curve-shaped reverse order diffraction profiles in Fig. α, the vertical grating equation is modified into:At normal incidence of light  over total incident power (P0)).The superposition of Eq. λ) and low angular dispersion properties. The curved surfaces of the foil grating accumulate higher numbers of grooves under specific illumination conditions, in contrast to the flat grating. As the resolving power of a diffraction grating is proportional to the illuminated number of grooves and the periodicity30, the microscopic shape provides an advantage for achieving high resolving power. To be precise, the microscopic triangular shape increases the number of grooves by a factor of cosec(α/2). That results in two times the number of effective grooves when α = 60° for a fixed illumination spot compared to a flat grating with the same period. Moreover, according to Eq. m and period dv by a factor of 2cos(α/2). This reduces the angular spread of a spectrum of any order m. Therefore, the smaller angular spread of the foil grating enhances its “degree of iridescence”  compared to regular binary phase gratings.The detailed fine features in both the experimental and simulated scattering profiles of the foil grating are clearly evident in Fig. f cosecα/. That reWe further derived the vertical grating equation for the biomimetic foil grating from Eq.  by approα/2), α = 56°, the top angle of the foil prototype] as a conventional 2D grating of the same period (flat). Hyperspectral analyses show that natural spider scales , the diffraction pattern is dominated by the vertical nanograting. We note that the banded pattern shown in the scatterograms of the prism  of the microscopic grating, the angular separation between adjacent diffraction orders and the free spectral range of each individual order is small  and granular depositions were observed in the TEM micrographs of the scales  likely possess tetrachromatic colour vision , so female peacock spiders are likely able to perceive the displayed colours in the form of dynamic rainbow iridescence. Male peacock spiders court females at a distance of a few centimetres, and raise and wiggle their abdomens to display the abdominal colours to the females  for applications where fine-scale spectral resolution is required in a very small package, notably instruments on space missions, or wearable chemical detection systems. Therefore, a miniature spectrometer and light dispersive components will have significant impact to fields ranging from life sciences and biotechnology to material sciences and engineering.Advanced computer-aided design allows construction of optical components that have a complex and previously unimaginable geometry/topology with novel functionality, high efficiency, and a compact footprintM. robinsoni specimens were collected on 22 October 2012 by Peter Robinson at the type locality near Newcastle, New South Wales 47. M. chrysomelas  were collected by Jürgen Otto on October 2013 at several locations near Esperance in southwestern Western Australia47. M. chrysomelas is widespread in Australia, occurring in the east, west, central parts and the tropical north48. All specimens were preserved in 70% ethanol. Details of both species' distribution can be found in references47.TM UV–visible-NIR microspectrophotometer  with a 50× glass objective lens  = 0.7, free working distance = 1.1 mm, EC Epiplan 442060-9900-000, Carl Zeiss Microscopy, LLC.) for better brightfield colour imaging with extreme chromatic correction.Specimens were observed using a 20/30 PVTo investigate the surface morphology of peacock spider scales, opisthosoma cuticle fragments were attached to sample stubs using carbon tape. Spider samples were sputter-coated with gold-palladium for 3 min under 20 mA, 1.4 kV and observed under a scanning electron microscope  with 8 mm operating distance and 5 kV accelerating voltage.8. The cured epoxy block was trimmed with a Leica EM TRIM2 (Leica Microsystems) and microtomed into 80 nm thin sections using Leica EM UC6 (Leica Microsystems) with a DiATOME diamond knife . Sections were mounted onto 100 mesh copper grids  and observed under a transmission electron microscope  without sample contrast staining for visualization.Opisthosoma cuticle fragments were dehydrated, and washed, followed by epoxy resin infiltration and embedding based on previously reported protocol(s)nr = 1.52, IP-Dip, Nanoscribe GmbH) was drop-casted on a silicon wafer (500 μm thick with an oxidation layer of 3000 Å) and the objective lens immersed directly in the photoresist. A femtosecond laser  was used as the irradiation source. A laser power of 25 mW was used in the TPP process and was controlled by an acousto-optic modulator. 50 mm/s writing speed was controlled by a galvo-mirror scanner43. Each design was fabricated to a 135 × 135 μm2 area. The fabricated structures were then characterized using a Hitachi S-4700 SEM , sputter-coated with 5 nm of chromium. The imaging voltage was kept low (<10 kV) to avoid damaging the structures.We used two-photon polymerization (TPP) nanolithography to fabricate the designed engineering prototypes  for experimental investigations. The 3D laser lithography system  utilized a dip-in configuration with a 63×, 1.4 N.A. oil immersion objective lens  to focus the laser beam. An acrylic-based monomer liquid photoresist optimized for TPP applications  was used to provide spatial mapping of the spectral output from the samples when normally illuminated with white light. Each sample was imaged without a coverslip for structure and spectral mapping. For structure 20% of the light output was imaged using a monochrome QIClick camera (QImaging); for spectral reflectance measurements, 80% of the light output was collected using a 100× 0.9 N.A. air objective  on a Nikon Eclipse 80i microscope with a PARISS® spectrometer utilizing a Retiga 2000DC CCD camera (QImaging). A 50 µm slit was used for window collection, and radiometric calibration was done with a Hg+/Ar+ lamp (LightForm Inc.), with spectral resolution measured better than 2 nm. A silver mirror (Thorlabs) was used as a reflectance reference for all measurements. Hyperspectral mapping was performed using a library of selected spectra with a minimum correlation coefficient (MCC) of 0.99 used as a discrimination factor to identify and map common spectra. All spectra were smoothed, normalized, and plotted using GraphPad Prism statistical software . The colours of the curves were estimated based on the “spec2rgb” function in R script “pavo”49.A PARISS50. Scatterograms were obtained by focusing a white light beam with narrow aperture (< 5°) onto at a small circular area (diameter ~ 13 µm) of the object, and the spatial distribution of the far-field scattered light was then monitored.The spatial reflection characteristics of the iridescent spider scales and 3D printed grating designs were studied with an imaging scatterometer to visualize the diffraction patterns that they produced. The samples were glued to the tip of a glass micropipette, thus allowing us to position them in the first focal point of the ellipsoidal mirror of the imaging scatterometer® was applied to individual geometry that consists of one unit cell of the gratings . The Electromagnetic Waves, Frequency Domain interface of the commercial simulator COMSOL Multiphysicsngs Fig. a-d51. Dised Fig. . This coThe top and the bottom of the simulation unit cell were bounded by perfectly matched layers (PML). These boundary layers absorb any incident waves, preventing artifacts that could result from spurious interferences with re-entrant waves. Port boundary conditions were used to release the incident wave and to absorb the zeroth order (specular) reflected and transmitted waves. The input to each periodic port was an electric field amplitude vector with defined unit intensity. To assess angle-dependent scattering, the diffracting structures were rotated from −60° to 60° keeping the angle of light incidence at normal and zeroth order reflectance was calculated using S-parameters for wavelengths of 400–700 nm.All relevant data are available from the authors upon request.Supplementary InformationPeer Review File


In this review, we summarise and critically evaluate the physiological and performance effects of dietary NO3− supplementation during exposure to simulated and terrestrial altitude. Previous investigations at simulated altitude indicate that NO3− supplementation may reduce the oxygen cost of exercise, elevate arterial and tissue oxygen saturation, improve muscle metabolic function, and enhance exercise capacity/performance. Conversely, current evidence suggests that NO3− supplementation does not augment the training response at simulated altitude. Few studies have evaluated the effects of NO3− at terrestrial altitude. Current evidence indicates potential improvements in endothelial function at terrestrial altitude following NO3− supplementation. No effects of NO3− supplementation have been observed on oxygen consumption or arterial oxygen saturation at terrestrial altitude, although further research is warranted. Limitations of the present body of literature are discussed, and directions for future research are provided.Exposure to altitude results in multiple physiological consequences. These include, but are not limited to, a reduced maximal oxygen consumption, drop in arterial oxygen saturation, and increase in muscle metabolic perturbations at a fixed sub-maximal work rate. Exercise capacity during fixed work rate or incremental exercise and time-trial performance are also impaired at altitude relative to sea level. Recently, dietary nitrate (NO Exposure to this hypoxic environment decreases the amount of oxygen (O2) reaching the lungs, blood, and ultimately tissue  (square brackets denote concentration)   , muscle lactate]  are typineration  and/or mneration .3− supplementation attenuated intramuscular phosphocreatine (PCr), adenosine diphosphate (ADP), and inorganic phosphate (Pi) perturbations and lowered 2+) handling , [Pi], and pH as assessed via 31phosphorus-magnetic resonance spectroscopy (31P-MRS) during leg extension exercise in normobaric hypoxia  . It was speculated that this might be related to reversible S-nitrosation of creatine kinase (CK), and could serve to enhance mitochondrial sensitivity to ADP stimulation. Thus, NO3− supplementation may enhance both muscle energetics and O2 delivery during hypoxic exercise, which might partly underpin the improvements in exercise performance discussed in Sect. NO~2800 m) . Muscle ng (MRI) . The T2*ng (MRI) . The autneration . NO3− srgy (∆G) . Interes3− supplementation has been reported to reduce BP  had returned to baseline values. This suggests possible storage and metabolism of NO2−, and NO in the extravascular tissue could contribute to hypoxia-induced vasodilation  age 25 ± 1 years) exercising in normoxia and acute normobaric hypoxia (FIO2 titrated to achieve ~80% SaO2) [2−], NO3− supplementation significantly increased the vasodilation and blood flow response to hypoxic exercise in older participants (mean ± SD age 64 ± 2 years). The magnitude of the effect was such that age-related differences in compensatory vasodilation were abolished with NO3− supplementation, suggesting therapeutic utility of NO3− in older populations exercising in hypoxia ) in normobaric hypoxia . Improvements in −1 blood [lactate]) and average power output during a 30-min TT in normoxia were similar in NO3− and placebo conditions. Likewise, the pre- to post-hypoxic training change was similar between groups for muscle adenosine monophosphate-activated kinase (AMPK) protein content and phosphorylation, hypoxia-inducible factor (HIF)-1α messenger RNA (mRNA) content, and glycogen breakdown during the TT. More recently, De Smet et al. [3− on adaptations to sprint interval training (SIT) in normobaric hypoxia  versus separate unsupplemented normoxic and hypoxic training groups. Participants completed 3 × 4–6 maximal 30 s cycle ergometry sprints per week over a 5-week period. The proportion of type IIa fibers was significantly increased in the hypoxic NO3− group, whereas the proportion of type IIx fibers was significantly decreased. By contrast, a significant decrease in the proportion of type IIx fibers in the unsupplemented groups did not lead to significant increases in other fiber-type groups. These findings were coupled with a tendency towards greater improvements in normoxic 30-s sprint performance in the hypoxic NO3− training group relative to the hypoxic unsupplemented training group. Conversely, performance in a 30-min TT completed in normoxia increased by similar amounts in all groups. Taken together, the results of Puype et al. [3− supplementation consumed during 5–6 weeks of endurance or sprint training in hypoxia has minimal effects on physiological adaptations or on exercise performance in normoxia.Puype et al.  found not et al.  explorede et al.  and De Se et al.  suggest 3− supplementation on the adaptations to SIT in normoxia [3− versus placebo prior to SIT sessions . Thompson et al. [3− compared with both training individuals given a placebo and NO3−-supplemented individuals who did not train. Interestingly, and supporting the findings of De Smet et al. [3− but not in the other conditions. Based on the findings of these studies, it is possible that NO3− may elicit similar muscle fiber-type changes when consumed parallel to training in normoxia and hypoxia but may be more effective at enhancing performance adaptations to normoxic than hypoxic training. However, this requires direct investigation given the multifarious methodological differences between studies. In particular, Puype et al. [−1·min−1), whereas Muggeridge et al. [−1·min−1), who may exhibit a greater response to training with NO3−.Two studies have also recently explored the effects of NOnormoxia , 141. Munormoxia  reportedt et al. , Thompsoe et al.  and De Se et al.  in hypoxe et al.  and Thom3− supplements  in an attempt to maximize performance in some, but not all, training sessions. This approach, first highlighted by Professor Andrew Jones of Exeter University, UK [3− supplementation in hypoxia has yet to be evaluated scientifically but may offer valuable psychological advantages to athletes regardless of the potential physiological merits.The approach adopted in the above studies is similar to most training-intervention studies, whereby a standardized exercise session is utilized throughout the intervention, either with or without an overload component –145. Thisity, UK , 32, maysity, UK , 32. Per3− supplementation is emerging as a promising nutritional aid, with potentially beneficial applications for the wide variety of individuals ascending to altitude each year. In this review, NO3− supplementation has been demonstrated to reduce pulmonary aO2 in normobaric hypoxia—effects that may be attributable to improvements in the efficiency of muscle contraction and/or mitochondrial respiration, and are of functional relevance for individuals exercising in a low-O2 environment. Current evidence also suggests that NO3− supplementation can improve muscle energetics during exercise in normobaric hypoxia via effects on tissue O2 delivery and consumption and might alter cardiovascular responses to normobaric and hypobaric hypoxia. In contrast, whilst NO3− supplementation might influence some of the physiological responses to hypoxic training, current evidence suggests that this does not translate into improved exercise performance in normoxia. Given that the majority of published investigations have explored the effects of NO3− supplementation in simulated altitude, which is often regarded as an incomplete surrogate of “true” altitude, future studies at terrestrial altitude are necessary. Researchers are encouraged to further probe the myriad potentially beneficial effects of NO3− supplementation as a potential aid to “beet-ing” the mountain.NO


INa, based on 30 year old data from chick embryo cell aggregates). Here we develop a simple “parsimonious” rabbit AP model that is mathematically identifiable  by combining a novel Hodgkin-Huxley formulation of INa with a phenomenological model of repolarization similar to the voltage dependent, time-independent rectifying outward potassium current (IK). The model was calibrated using the following experimental data sets measured from the same species (rabbit) under physiological conditions: dynamic current-voltage (I-V) relationships during the AP upstroke; rapid recovery of AP excitability during the relative refractory period; and steady-state INa inactivation via voltage clamp. Simulations reproduced several important “emergent” phenomena including cellular alternans at rates > 250 bpm as observed in rabbit myocytes, reentrant spiral waves as observed on the surface of the rabbit heart, and spiral wave breakup. Model variants were studied which elucidated the minimal requirements for alternans and spiral wave break up, namely the kinetics of INa inactivation and the non-linear rectification of IK.The simplicity of the model, and the fact that its parameters have physiological meaning, make it ideal for engendering generalizable mechanistic insight and should provide a solid “building-block” to generate more detailed ionic models to represent complex rabbit electrophysiology.Elucidating the underlying mechanisms of fatal cardiac arrhythmias requires a tight integration of electrophysiological experiments, models, and theory. Existing models of transmembrane action potential (AP) are complex (resulting in over parameterization) and varied (leading to dissimilar predictions). Thus, simpler models are needed to elucidate the “minimal physiological requirements” to reproduce significant observable phenomena using as few parameters as possible. Moreover, models have been derived from experimental studies from a variety of species under a range of environmental conditions  under nearly identical physiological conditions, we developed a simple model of the electrical activity of a cardiac cell derived from sub-cellular, cellular, and tissue experimental data. This model reproduces cellular excitability and its recovery as well as several important “emergent” phenomena including beat-to-beat cellular alterations and unstable spiral waves. Under some conditions, unstable spiral waves in this model give rise to continuous formation of new spiral waves  which is thought to be the underlying cause of cardiac fibrillation and sudden cardiac death. The simulation of action potentials (APs) and their propagation has been an integral part of the field of electrophysiology for over half a century. Over a hAn alternative, and complementary approach compared to these ionic models, is to use “phenomenological” models which are designed to represent specific, often macroscopic phenomena –19. PhenINa) and the rectifying potassium current (IK1), which are both highly non-linear functions of transmembrane potential (Vm). INa is responsible for the rapid all-or-none depolarization of the AP upstroke, and IK1 is responsible for maintaining the resting potential near -85 mV. In fact, it has been shown recently that transfecting cells with genes encoding for cardiac INa, IK1, and gap junctions Cx43 (only) into unexcitable somatic cells can transform monolayers into excitable tissue capable of supporting propagating “cardiac” action potentials including reentrant “spiral” waves![The two most important membrane currents for cardiac excitability are the rapid sodium current  data from spherical clusters of eleven day-old embryonic chick heart cells,[INa model based on rabbit data, but this is not a simple task. To date, INa activation parameters have been derived exclusively from VC protocols; however, since the INa current in the adult myocyte is too large to allow adequate voltage control under physiological conditions,[INa channel (Nav 1.5).All existing ionic models of the rabbit ventricular AP –27 incorig model, albeit wrt cells, and slowrt cells, Obviouslnditions, these VConly on data recorded from the rabbit under physiological conditions. A model is considered parsimonious if it accomplishes a desired level of explanation or prediction with as few parameters as possible . For this paper, the desired level of prediction is the following well-known and important electro-physiological phenomena, measured from rabbit ventricular myocytes/tissue under nearly identical and physiological conditions: 1) steady-state inactivation as determined from voltage clamp experiments [INa model with various models of IK1, and also explored a range of cellular and tissue behavior in an AP model incorporating a previously published phenomenological two parameter model of repolarization.[The first aim of this paper is to develop a “parsimonious” model for the rabbit myocyte AP based eriments ; 2) actieriments ; and 4) eriments  For calirization. The overINa are of the form pioneered by Hodgkin-Huxley (HH) [Vm is the transmembrane potential, gNa is the maximal conductance of INa, m (fast activation) are gating variables, and h (fast inactivation), and ENa is the Nernst equilibrium potential for sodium. Beeler and Reuter [j)INa models.[The equations of ley (HH) INa=gNamd Reuter  introduca models.–27.y represents each gating variable, αy(Vm) and βy(Vm) represent the on and off rate constants for gating (respectively) which are voltage dependent, y∞(Vm) and τy(Vm) are the steady state fraction of activation or inactivation and time constant (respectively) and are functions of Vm. Hodgkin and Huxley [αy(Vm) and βy(Vm) using only 9 parameters; in contrast the LR1 INa equations contain 31 parameters. Since 1952 there has been significant advancement in the derivation of gating equations based on first principles.[δy ≤ 1 are positive, while ky < 0 for activation and ky > 0 for inactivation. Using y∞(Vm) and τy(Vm) from Ey,ky,δy) in this formulization. The main limitation of this formulization is that τm(Vm)→0 away from Em which is not realistic, because gating transformations cannot be instantaneous. For example, the LR1 equations predict τm(−85mV) = 0.0055 ms and τm(+30mV) = 0.041 ms at 37 C, however these values are below the resolution of voltage clamp measurements. We address this problem by removing the Vm dependence of τm . To summarize, we define the novel PR INa submodel using Eqs m∞ and h∞ given by τh given by τm equal to a constant.The HH equations for the gating variables are of the form:dydt=αy−y−βyy≡y∞inciples., 36 The inciples.αy=αy0exdydt=αy−y−βyy≡y∞Table 1. Values for Eh and kh where taken directly from Table 1 in [ENa = 65mV, and all other parameters were determined via manual fitting to the experimental data described below using no more than 2 significant digits; δh were computed via “inverting” τh(−80mV) = 4.0ms and τh(0mV) = 0.45ms.All model parameters are provided in ble 1 in . We set Vm with dt = 0.001 ms which is actually a very conservative choice made because of the low computational cost. The fact that τm is constant provides for a well-defined minimum time constant of the model which allows for the use of larger values of dt and computational efficiency compared to other models. [Iion = INa + IK1L  with diffusion coefficient (D) equal to 0.001 cm2/ms and dx = 0.01 cm, where IK1L is the formulation of IK1 provided by Livshitz and Rudy [INa and Vm signals were recorded at the center of the cable to construct the I-V curves for calibration to the experimental data as described below.Simulations required for the calibration were carried out in single cells and a one-dimensional cable by integrating the three ordinary differential equations using exact integration for the gating variables and forward Euler integration for  models.  Cable siand Rudy . INa andINa, therefore the precise time course of Vm(t) contains a wealth of information regarding INa kinetics.[INa can be estimated as Cm is the specific membrane capacitance which we assume is equal to 1 μF/cm2. During propagation, however, the total transmembrane current is not proportional to INa as CV is the conduction velocity [Vm measurements during AP upstrokes from isolated rabbit ventricular myocytes[Vm traces from APs stimulated with near threshold stimuli with a latency of 2.5 ms (range of 1.2 to 3.7 ms) were chosen from a previous study[INa of near –40 mV. Similar to Joyner et al. [The current during the AP upstroke is predominately kinetics.–42 In isvelocity . These r myocytes and from myocytes In isolaous study, becauser et al.  we foundcellular phenomenon [INa current and the AP shape. To reproduce the experimental results of recovery of AP excitability in isolated myocytes,[INa using APs measured during pacing from six isolated myoctes recorded in a previous study.[th beat the simulations were switched to current-clamp mode and stimulated with a 2 ms stimulus at various coupling intervals; the amplitude was adjusted such that the latency was 1 ms. The resulting peak INa was normalized to that recorded during the 10th beat and presented as a function of coupling interval.It is well known that enomenon . Here wemyocytes, we develus study. During tINa equations in all previous rabbit models are identical to LR1, except for scaling (conductances), we present previous model predictions using LR1 with two conductance values (23 mS/μF and 8 mS/μF) to cover the entire range (grey lines in figures). In addition we present results from the Ebihara-Johnson (EJ) model [Since the J) model   designed to reproduce rabbit AP repolarization during propagation:gK is the maximal conductance of IK, b is a parameter controlling AP shape, and EK is the reversal potential for potassium ; the nominal parameters values for IK are provided in Table 1, as previously determined.[INa + IK in Eqs Table 1.To create our parsimonious rabbit (PR) cell model, we combined our model of termined. We defin)(Vm−EK),where gKVm during AP depolarization of isolated rabbit ventricular myocytes are shown in Fig 1A; the traces are aligned to Fig 1B. The calibrated PR results are shown as a thick black line. Previous model predictions (grey lines) bracket the range of peak INa but do not accurately capture the initial inward current deflection of the experimental I-V relationship (see box).The experimental traces of propagation from [Fig 2A. Similar to above, previous model predictions (grey lines) span the range of peak INa but do not accurately capture the initial inward current deflection of the experimental I-V curve (see box). Although the PR model fits the initial decrease of INa and maximum Vm well, it does not match the experimental dynamic I-V curve during the second half of the upstroke.The experimental dynamic I-V curves during the AP upstroke during ion from  are showVm < -22 mV and for Vm > +4.5 mV.It is important to note that the experimental dynamic I-V curves for isolated myocytes  is diffeINa as a function of recovery time are shown in Fig 3B; experimental results from [Unlike previous models with slow inactivation , the PR The normalized magnitude of peak lts from  are showINa activation and inactivation as well as the corresponding time constants are shown in Fig 4 for our PR model, as well as LR1, and EJ models. Our PR model exhibits a steeper activation slope at slightly less depolarized voltages compared to LR1 and EJ (panel A). As described above, τm in the PR model does not exhibit voltage dependence but is similar to the mean value of LR1 and EJ (panel B). The steady state INa inactivation for PR is more negative compared to LR1 and EJ (panel C) and the maximum value of τh for PR is less than that for τh for LR1 and EJ80 and is considerably less than τj for LR1 (panel D).The voltage dependence of steady state Figs 2 represent the results from for our novel INasub-model only; the results in Fig 3 include coupling to the IK1L sub-model. To assess the effect of repolarization current on the PR results shown in Figs 3 we reran all simulations with cell models incorporating IK1L or the phenomenological model of repolarization INa sub-model only; in fact the results are shown as thick dashed (IK1L) and dotted (IK) lines in Figs 3 but cannot be seen because they superimpose upon the solid black line.The PR results in 2, APD = 197ms for single cells; and 2, CV = 55 cm / s and APD = 142ms for propagation in a cable. With the exception of APD, these values were not significantly altered (< 2%) compared to simulations using IK1L [IK [INa sub-model are quite insensitive to the specific choice of repolarization current.Simulations using our PR cell model resulted in APs with the following characteristics: ing IK1L  or IK [3INa gating parameters on eight quantities of interest; results are provided in the Table A in INa + IK) by varying repolarization parameters gK and b over the entire “physiological range” for all mammals: reported values of gK range from 0.1 to 0.5 mS/μF and the range of b values chosen, 0.03 ≤ b ≤ 0.05 mV-1, correspond to APDs ranging from 23 ms to 516 ms. The results are displayed in the Figs B and C in gK and b, IK parameters: CV: 4.5%. The fact that action potential depolarization characteristics are fairly insensitive to repolarization in single cells and during propagation provides support for the robustness of our INamodel.We performed parameter sensitivity analysis by quantifying the effect of a 1% variation of the eight dVm/dt)max, and CV.To confirm that the data used in the model calibration is representative, we compared several model results to experimental data from previously published studies. Table B in Fig 5. The cycle length (~ 150 ms) and line of block (~ 3.2 cm) for PR using the nominal parameter values were larger than the experiments. Incidentally, decreasing parameter b by only 4% decrease improved the correspondence considerably . This change is justified because the PR model does not include any repolarization kinetics and is not developed to reproduce APD shortening with rate. In contrast to the experimental results of Schalij et al., [Fig 5B consistent with previous results indicating SWB for this model in a 7.5 cm x 7.5 cm region.In addition, we compared the predictions of spiral waves in the PR and Mahajan (M08) models (5 cm x 5 cm) to those recorded in the whole rabbit heart by Schalij et al. Schalij  et al.,  the linelock ~ 3. cm for PThe level of validation required for a model depends on its “context of use” and the consequences of incorrect model predictions. Hence finterdependent. As shown in Fig 6, the dynamics of spiral waves in our model varied considerably as a function of repolarization . Spiral waves were not stable  for any parameter pairs  in the range studied. For 8 of 9 parameter pairs, the spiral wave tip followed typical non-circular trajectories, while spiral wave breakup (SWB) occurred for one parameter set . The frequency of activity for single spiral waves ranged from ~3 Hz to ~18 Hz and the frequency of activity during SWB was ~30 Hz.During “functional” spiral wave reentry the depolarization and repolarization processes are APD = 23ms which is not consistent with normal rabbit physiology) we investigated whether cellular alternans occurred in our PR model. We paced the PR cell model with a 2 ms stimuli of -20 pA/pF and found that alternans occurred for cycle lengths between 202 and 210 ms, as shown in Fig 7, which is within the range (190–240 ms) reported previously for isolated rabbit myocytes.[APD alternans occurred as a result of alternations of peak INa, which resulted in alternans in APD. Since INa is negligible except during depolarization, APD is a function of only h were more pronounced than m , we hypothesized that only INainactivation kinetics are necessary for cellular alternans. A “reduced” two variable  model, in which variable m3 was replaced with function gNa was reduced to 5.8 mS/μF (to maintain CV = 55cm / s), confirmed that INainactivation by itself was capable of generating cellular alternans in our PR model.Since SWB was observed , and therefore, IK parameters completely determines AP shape [n > 1 components, the total number of possible interactions between two or more components, excluding the full model of all n components, is 2n − n − 2. Traditional models have at least 10 components, which results in over 1000 interactions! Characterizing these interactions appropriately both physiologically and mathematically continues to be a significant challenge.Physiological models are developed using a set of experimental calibration data, which for our model is for rabbit tissue and myocytes at physiological temperature and heart rate. Our approach should be relatively straight-forward to implement for other species, although this will require the high-fidelity measurement of the dynamic I-V curve during propagation  and in sAP shape , in our AP shape . In addiphysiological insight regarding the interaction of depolarization and repolarization processes. We found that IK conductance (gK) is related to the cycle length of reentry  exhibited important “emergent” behavior including cellular alternans  resulted in a rate (~30 Hz) which is twice that observed in experiments.[Surprisingly, our PR model  at 0 mV for 2 ms.While our PR model reproduces important physiologically meaningful behavior, it does have limitations. It should be noted that the alternans resulting from the interaction of obtained  in the pobtained . The INakinetics. ImportanINa sub-model and previous rabbit INa sub-models is that we did not include a slow inactivation gate. While slow inactivation of INa can be justified from voltage clamp experiments [INa sub-model in the existing rabbit models are the same as LR1 in which the slow inactivation kinetics were derived from sheep and calf trabeculae muscles in which spatial homogeneity of Vm and VC control are lacking. We believe that much more research is needed to appropriately incorporate slow inactivation of INa (as well as late INa) into AP models, especially those used to simulate propagation. Incidentally, ten Tusscher et al. interpolated experimental time constants of fast and slow activation as a function of voltage over the range -80 to -40 mV in which data is not available to values more than four times values outside of this range .INa,IK) and three variables  are required to reproduce cellular alternans and spiral wave breakup. In fact, only two variables  are required to produce alternans and implicate a destabilizing effect resulting from the interaction of the non-linear rectification of IK [h. We believe that our PR model is an ideal tool to quantify excitability and propagation related to a variety of important physiological and pathophysiological issues. For example our model can be used to study the effects of drugs (our model predicts that an 83% decrease of gNa is required for propagation failure), genetic mutations of SCN5A such as LQT3, and the differences between atrial and ventricular tissue. In addition we believe that it provides a solid “building-block” to develop more realistic rabbit AP models by including additional sub-models .Our novel PR model provides unique and physiological generalizable insight into the relationship among gating kinetics and AP behavior in single cells and during propagation. We show unequivocally that only two currents  will be made available online at Research Gate. The model will be uploaded to CellML.S1 TextSupplementary information including Tables A and B as well as Figs A, B and C.(DOCX)Click here for additional data file.S1 Video(AVI)Click here for additional data file.S2 Video(AVI)Click here for additional data file.


Arterial oxygen partial pressure can increase during inspiration and decrease during expiration in the presence of a variable shunt fraction, such as with cyclical atelectasis, but it is generally presumed to remain constant within a respiratory cycle in the healthy lung. We measured arterial oxygen partial pressure continuously with a fast intra-vascular sensor in the carotid artery of anaesthetized, mechanically ventilated pigs, without lung injury. Here we demonstrate that arterial oxygen partial pressure shows respiratory oscillations in the uninjured pig lung, in the absence of cyclical atelectasis (as determined with dynamic computed tomography), with oscillation amplitudes that exceeded 50 mmHg, depending on the conditions of mechanical ventilation. These arterial oxygen partial pressure respiratory oscillations can be modelled from a single alveolar compartment and a constant oxygen uptake, without the requirement for an increased shunt fraction during expiration. Our results are likely to contribute to the interpretation of arterial oxygen respiratory oscillations observed during mechanical ventilation in the acute respiratory distress syndrome. Respiratory PaO2 oscillations smaller than 16 mmHg have been detected with slow response time sensors2 in animals with uninjured lungs when abnormally large tidal volumes (VT) greater than 20 ml kg−1 or even 30 ml kg−1 were delivered during mechanical ventilation4.In the healthy lung, arterial partial pressure of oxygen , causing PaO2 to increase during inspiration and decrease during expiration. This phenomenon may be used to personalize mechanical ventilation based on real-time, continuous PaO2 monitoring, where the appearance of an oscillating PaO2 signal may be a diagnostic biomarker for cyclical atelectasis.In the injured lung, especially in animal models of the acute respiratory distress syndrome (ARDS), large PaO2 oscillations observed in animal models of ARDS mostly appeared in association with a high average VT of 20 ml kg−1 13, 30 ml kg−1 14, or greater10. These large VT were delivered in experiments without application of positive end-expiratory pressure (PEEP), and imposing elevated peak end-expiratory pressures in order to provoke cyclical atelectasis in the injured lung. Even with these large VT, PaO2 oscillations only between 3 and 22 mmHg were observed in the uninjured lung, at a RR lower than 10 breaths per minute, and mean PaO2 above 450 mmHg15.A significant limitation of the above mentioned studies is that the PaO2 levels. Experimental evidence for this response to mechanical ventilation could also be useful to understand response of the lung to mechanical ventilation in the presence of injury.No study employing fast oxygen-sensing technology has explored the responses of the uninjured lung to mechanical ventilation at physiological PaO16, optimized its response time and linearity20, tested it in vitro21, showed it to be resistant to clotting, and demonstrated it to be capable of detecting rapid PaO2 changes in an animal model of ARDS34. With this new technique to study gas exchange dynamically, we explore here PaO2 responses to mechanical ventilation in a porcine model.We developed a fibre optic oxygen sensor2 declines at a rate that is inversely proportional to lung volume, as had been modelled23, and that this decline would be readily measurable in the uninjured pig lung during 20 s end-expiratory and end-inspiratory breath holds. We also hypothesised that, during tidal breathing, PaO2 oscillates dynamically in phase with lung volume in the uninjured lung, in the absence of cyclical atelectasis. This hypothesis led us to also explore the possibility that different ventilation strategies (pressure and volume control) and inspired-to-expired ratios could affect the mean PaO2 value, while airway pressures (end-expiratory and end-inspiratory), respiratory rate, or fraction of inspired oxygen (FIO2) were kept unvaried.We hypothesised that in apnoea or expiration PaO2 as observed in an anesthetised pig with uninjured lungs during volume (VC) and pressure control (PC) mechanical ventilation. PaO2 oscillations with an amplitude of about 10 mmHg and the same frequency as breathing were observed at a RR of 12 breaths per minute, inspired-to-expired ratio (I:E) of 1:1, tidal volume (VT) of about 10 ml kg−1, and at mean PaO2 near 130 mmHg. The peak airway pressure was maintained below 13.6 cmH2O. The airway pressure remained lower than the mean pulmonary artery pressure throughout the experiment.Figure 2 decline over time during breath hold manoeuvres at end-expiratory volume , end-inspiratory volume , and at the end of a large inspiration . The figure illustrates the slower rate of decline observed at the larger lung volume, where also a transient increase in PaO2 was observed immediately following the large inspiration. There was good agreement between measurements of VT recorded via the respiratory monitor (Datex Ultima) and estimates of the lung volume increases consequent upon inflation made by CT image analysis: the mean volume difference (i.e. bias) was −22 ml, with limits of agreement between −67 (95% CI −80 to −54) and 22 (95% CI 9 to 35). The SD of the differences was 23 ml (SE ± 3.7 ml)  Figure . Represep < 0.001), as were the associated rates of PaO2 decline (p < 0.01). The rates of PaO2 decline ranged from 6.9 ± 1.3 mmHg s−1 at end-expiratory lung volume to 3.4 ± 0.6 mmHg s−1 during the large inspiration (VT20). Table Table 2 values over time during volume (A–D) and pressure (E–H) control mechanical ventilation delivered at a RR of 12 breaths per minute, with PEEP set at 5 cmH2O. These two ventilation modes and I:E ratios of  1:2,  2:1,  1:4 and  4:1 affected both the mean PaO2 and the oscillation amplitude. Similarly, Fig. 2 values over time for a RR of 6 breaths per minute, where greater changes in mean PaO2 levels and larger oscillations were observed.Figure 2 values associated with the eight combinations of mechanical ventilation settings studied at RR of 12 (VT = 10 ml kg−1), and the associated mean airway pressure. In the experiments considering I:E of 1:2 and 2:1, the ventilation mode (pressure or volume control) resulted in significant PaO2 changes , as did inverting I:E ratios (i. e. switching the duration of inspiration with that of expiration) ; no significant interaction effect was observed [p > 0.05]. In the experiments considering I:E of 1:4 and 4:1, the ventilation mode resulted in significantly greater PaO2 changes , as did inverting I:E ratios , with a significant interaction . The greatest mean PaO2 change was observed when I:E was inverted from 1:4 in volume control mode to 4:1 in pressure control mode, when the mean PaO2 increased by 19 mmHg, from 125 mmHg (±10 mmHg) to 144 mmHg (±8 mmHg). At RR12 the amplitude of the PaO2 oscillations decreased on average from approximately 10 to about 7 mmHg when inverse-ratio ventilation was used. The reduction in amplitude of the PaO2 oscillations was particularly clear at RR6 (VT = 20 ml kg−1), when inverse-ratio ventilation reduced average PaO2 oscillations from 40 to 15 mmHg, observed respectively in pressure control mode (I:E 1:4) and volume control mode (I:E 4:1). Table 2 change was observed when I:E was inverted from 1:4 in volume control mode to 4:1 in pressure control mode, when mean PaO2 increased by 24 mmHg, from 137 mmHg (±18 mmHg) to 161 mmHg (±18 mmHg).Table The change in the mass of the slice that was atelectatic was less than 2% C.I. 1.5–2.0%] during tidal breathing. Figure –2.0% durFigure 2. These simulations showed changes in mean PaO2, and PaO2 oscillation amplitudes that were similar to those observed in vivo under the various conditions imposed.Figure 2 declined at different rates during breath hold manoeuvres depending on lung volume in vivo, that significant dynamic PaO2 oscillations occur, and that these PaO2 oscillations are determined by alveolar oxygen changes within the respiratory cycle.In this experimental study in mechanically ventilated pigs with uninjured lungs, we showed that PaO2 oscillations were observed at respiratory rates of 12 and 6 breaths per minute, at mean PaO2 values between ~120 and ~150 mmHg, at any I:E ratio and ventilation mode considered in this study. These PaO2 oscillations achieved maximum amplitudes greater than 50 mmHg in the absence of cyclical atelectasis as measured by CT image analysis during ventilation in pressure control mode at a RR of 6 breaths per minute, I:E 1:4, VT of 20 ml kg−1, and with 5 cmH2O PEEP. In this sense, any shunt should be of the same magnitude throughout the respiratory cycle, unless blood flow is redistributed within the breath. The observed increase in PaO2 during inspiration suggests that this redistribution of blood flow to non- or poorly ventilated regions did not occur, indicating that it was not a determinant of respiratory PaO2 oscillations.During tidal breathing with mechanical ventilation, PaO2 value, as observed by altering I:E ratios and ventilation control mode. In particular, mean PaO2 was higher when inspiration lasted longer than expiration, which is when lung volume is greater across the respiratory cycle. Moreover, the amplitude of the PaO2 oscillations was smaller at greater mean lung volume, suggesting that there was a slower PaO2 decline during the expiratory phase at I:E of 2:1 and 4:1 than when these I:E ratios were inverted. Overall, these respiratory PaO2 oscillations could be predicted from the oscillations in arterial partial pressure of carbon dioxide24.In agreement with the results obtained during breath holds, the weighted-average lung volume was a strong determinant of mean PaO2 oscillations have previously been interpreted primarily as being caused by cyclical atelectasis associated with tidal recruitment and derecruitment during ventilation in animal models of the acute respiratory distress syndrome (ARDS)25. Differences between our work and the published literature include animal species, smaller tidal volumes, greater RR, reduced airway pressures, physiological mean PaO2, and crucially that in our work we studied uninjured lung as opposed to animal models of lung injury. It is possible that the use of much larger tidal volumes (as it appears in the literature) might have resulted in larger PaO2 oscillations in our uninjured lung model; we did not investigate this possibility as tidal volumes greater than those studied in our experiments are neither physiological, nor of clinical interest26. It is intriguing to notice that the PaO2 oscillation amplitude of ~22 mmHg observed at I:E of 4:1, RR 6, VT 20 ml kg−1, mean PaO2 value of ~161 mmHg in our work is similar to that of ~25 mmHg reported in a pig saline lavage model of ARDS in rather similar conditions 27. In this respect, it is possible that the oscillations reported in the literature were caused in part by oxygen mass balance as described in equations 4 and 5 (ventilatory delivery vs pulmonary uptake) rather than cyclical recruitment and derecruitment.Respiratory PaO2 rate of decline was associated with lung volume, we performed a series of breath hold experiments at EELV with 5 cmH2O, end-inspiration and at the end of a large inspiration. We showed that PaO2 declined more rapidly at smaller lung volumes than it did at larger lung volumes. This result is in agreement with an original observation performed with slower oxygen saturation sensors during apnea, when the rate of saturation decline was “inversely proportional to the volume of air in the lung during apnea and closely related to the oxygen saturation at the beginning of apnea”28.To emphasize the observation that PaO2 decline could be partly explained by lung recruitment and derecruitment in anaesthetized animals. Lung recruitment could be observed during end-inspiratory breath holds, especially during a breath hold performed after a large inspiration, and derecruitment could be observed during an end-expiratory breath hold. Lung recruitment during an end-inspiratory breath hold could reduce the rate of PaO2 decline, and vice versa during an end-expiratory breath hold. However, in this study, we were unable to detect atelectatic mass changes during breath holds using CT image analysis, which did not provide any evidence for recruitment or derecruitment during end-inspiratory or end-expiratory breath holds respectively. Indeed, this result was partly expected given the short duration of the breath hold manoeuvres, the relatively low pressures used during the inspiratory breath holds, the application of 5 cmH2O PEEP during the end-expiratory breath holds, and the lack of known lung injury or gross regional ventilation-perfusion mismatch.This difference in the rate of PaO2; in particular, elevated airway pressures applied to the lung during inspiration could cause a prevalence of zone 1 aeration30, where the non-dependent lung regions receive little or no perfusion also due to changes in lung blood volume31. This phenomenon per se would increase the rate of PaO2 decline during an end-inspiratory breath hold, due to a reduced oxygen uptake in the zone 1 regions of the pulmonary circulation. The highest airway pressure applied in our experiments was 24 cmH2O during breath holds at the end of a large inspiration, when the rate of PaO2 decline was slower than during end-inspiratory and end-expiratory breath holds. Importantly, the airway pressure did not exceed the pulmonary artery pressure at any time during breath holds, and overinflated lung mass was never greater than 0.1%, suggesting that zones 1 were not dominant, hence did not affect the rate of PaO2 decline.Ventilation and perfusion changes during a breath hold could determine real time changes in PaO2 change is caused by alterations in either ventilation, or perfusion, or both, and that it does not provide evidence for their spatial distribution within the lung. In our study, the dynamic CT image analysis partly compensated for this limitation by providing an index of lung aeration and atelectasis. Although this information was anatomically limited to a single slice during tidal breathing, the antero-posterior orientation of the slice could be representative for the effect of the gravitational gradient on pulmonary aeration32.Two main limitations of this technology are that it is unable to demonstrate whether a respiratory PaO2 oscillations to appear during mechanical ventilation. We conclude that the mechanism determining these respiratory PaO2 oscillations is the variation of alveolar oxygen tension within the breath, with oxygen entering the alveoli during each inspiration, and that these cyclic variations in alveolar oxygen tension are transmitted all the way to the systemic arteries. Our results in an anaesthetized, ventilated pig model, in the absence of lung injury, may have implications for the interpretation of the results from conditions of lung disease33, in particular from patients with lung injury, and from animal models of ARDS.Our study demonstrates that cyclical atelectasis is not necessary for respiratory PaOThe study was approved by the Animal Research Ethics Committee at Uppsala University and by the UK Home Office. All the animal experiments conformed to the National Institutes of Health Guidelines for the Use of Laboratory Animals. Relevant sections of the ARRIVE guidelines were followed. A total of 8 domestic pigs were studied (average ± SD: 29 ± 2 kg). Details regarding the anaesthesia, mechanical ventilation, instrumentation and measurements are reported in the supplementary information.2 was recorded continuously, simultaneously with analogue signals for cardiovascular and respiratory parameters from patient monitors.The intravascular, fibre optic oxygen sensor was inserted in a carotid artery via a standard arterial catheter, and PaO2 decline during apnoea, breath hold manoeuvres lasting at least 20 s were performed at three lung volumes: end-expiration , end of an inspiration corresponding to a VT of 10 ml kg−1 (VT10), and at the end of a large inspiration, corresponding to a VT of 20 ml kg−1 (VT20). Breath hold manoeuvres associated with these three lung volumes were performed six times each, in a sequence that covered each possible sequential effect, for a total of 18 breath hold manoeuvres per animal. The sequence in which breath hold manoeuvres were performed is presented in Table 2 decline was normalized to account for small differences in body weight between animals. The steepest rate of decline measured continuously over 5 s during each breath hold manoeuvre was considered for analysis. This period of 5 s was used to maximize the interval time while avoiding the inclusion of PaO2 values below 100 mmHg, when the rate of PaO2 decline decreases due to haemoglobin desaturation, which is beyond the scope of our study.In order to determine the rate of PaO2 and PaO2 oscillation amplitudes, and the potential effect of cyclical atelectasis, if present, in determining PaO2 during tidal breathing. We studied the effects of ventilation in pressure control mode, and also explored the effect of delivering the same VT gradually in volume control mode, recording PaO2 changes and determining oscillation amplitudes at the four I:E ratios. Airway pressure was monitored and controlled throughout the experiments. The two sets of tests started with either I:E of 1:2 or 1:4 in volume control mode achieving a mean PaO2 of about 130 mmHg, followed by a randomised sequence of any possible combination of I:E and pressure or volume control mode. Respiratory rates (RR) studied included 12 and 6 breaths per minute, with VT of 10 ml kg−1 and 20 ml kg−1 respectively. In total, we included 16 conditions . During tidal breathing, once steady state had been achieved, data collected over a period of 2 min were considered for analysis. Steady state was defined as mean PaO2 not changing by more than 5 mmHg min−1.We aimed to establish the effect of altering I:E ratios  on mean PaO2O was used throughout the experiments in order to prevent, or at least reduce the likelihood of collapse of dependent regions at end expiration.PEEP of 5 cmHWe used computed tomography (CT) to measure lung volumes as well as to estimate pulmonary atelectasis and its changes during the breath hold manoeuvres and dynamically during tidal breathing in two animals. Details of the CT images acquisition, analysis procedure, calculation of air volume and tissue mass are presented in the supplementary information.We hypothesised that the uninjured lung could be modelled as a single alveolar compartment with a constant oxygen uptake. The rate of change of oxygen in this compartment is equal to the difference in the rates of uptake by the pulmonary circulation and input via ventilation during inspiration, or elimination during expiration. Details of the mathematical modelling are presented in the supplementary information.2 data recorded during the breath hold manoeuvres were analysed by means of univariate repeated measures ANOVA with lung volumes as fixed factors , and post hoc analysis with the Bonferroni method. A two-way ANOVA was conducted to compare the main effects of ventilation mode and I:E ratio, and their interaction on mean PaO2 and its oscillations. Ventilation mode included two levels (volume and pressure control mode), and I:E ratio consisted of two levels . Values presented are mean ± standard deviation, unless otherwise stated. Significance level was accepted at p < 0.05.PaOData are available upon request.Supplementary Information


Here, reactive astrogliosis was induced with IL1β. Within 24 hours of IL1β induction, astrocytes acquired reactive characteristics. Transcriptome analysis of over 40000 transcripts of genes and analysis with PFSnet subnetwork revealed upregulation of chemokines and axonal permissive factors including FGF2, BDNF, and NGF. In addition, most genes regulating axonal inhibitory molecules, including ROBO1 and ROBO2 were downregulated. There was no increase in the gene expression of “Chondroitin Sulfate Proteoglycans” (CSPGs’) clusters. This suggests that reactive astrocytes may not be the main CSPG contributory factor in glial scar. PFSnet analysis also indicated an upregulation of “Axonal Guidance Signaling” pathway. Our result suggests that human spinal cord reactive astrocytes is potentially neuroprotective at an early onset of reactive astrogliosis.Reactive astrogliosis is a critical process in neuropathological conditions and neurotrauma. Although it has been suggested that it confers neuroprotective effects, the exact genomic mechanism has not been explored. The prevailing dogma of the role of astrogliosis in inhibition of axonal regeneration has been challenged by recent findings in rodent model’s spinal cord injury, demonstrating its neuroprotection and axonal regeneration properties. We examined whether their neuroprotective and axonal regeneration potentials can be identify in human spinal cord reactive astrocytes  The prevailing dogma of the involvement of reactive astrocytes in inhibition of axonal regeneration3, has been challenged by recent findings of Anderson et al. work, which demonstrated the critical role of reactive astrocytes in aiding axonal regeneration in rodents4. While some of the previous reports have identified reactive astrocytes as neuroprotective agents6, their direct neuroprotective effects were not well-documented in the human CNS astrocytes. As astrocytes’ functions differ among regions in the CNS7, we investigated the presence of axonal regenerating and neuroprotective properties of reactive astrocytes4 in human spinal cord derived astrocytes. Elucidating these properties at an early onset of reactive astrogliosis, will enable physicians and scientists to design novel therapeutic strategies exploiting the potential of reactive astrocyte mediated endogenous recovery.Reactive astrogliosis is a common response of astrocytes to most Central Nervous System (CNS) injury8. IL1β is a prominent inflammatory cytokine and is an early regulator of astrogliosis9. IL1β mRNA is highly up-regulated and persists 24+ hours after the onset of Spinal Cord Injury (SCI) at and around the epicenter of injury. This both initiates and modulates the inflammatory responses, leading to reactive astrogliosis10. Although bacterial endotoxin; lipopolysaccharides (LPS) could also be used for inducing reactive astrogliosis11, human astrocytes are unresponsive to LPS stimulation but are highly sensitive to IL1β12. In addition, it has also been reported that clusters of genes expression in reactive-astrocytes were highly dependent on various modalities of injury-induction14.It has been reported that among inflammatory mediators such as IL-1, TNF, IFN, and TGF, which are known to be active post-CNS injury, cytokine interleukin-1β (IL1β) is specifically critical for induction of reactive-astrocyte phenotypehuman spinal cord astrocytes, in vitro within 24 hours. Reactive astrogliosis is known to be invoked at the early phases of injury, with astrocytes acquiring a hypertrophic morphology and increased GFAP expression17  are the most up-regulated genes in human spinal cord reactive astrocytes. Whole genome transcriptome analysis shows changes in genes expression levels of 25 axonal growth permissive and 13 axonal growth inhibitory molecules. Particularly, the axonal growth promotion and neurotrophic factor genes like BDNF and NGF were upregulated. On the other hand, we detected no upregulation of CSPGs clusters of genes, which suggests that reactive astrocytes may not be the major contributors of CSPGs at the early onset (24 hours) of glial scarring. “Axonal Guidance Signaling” and “ECM-Receptor Interaction” pathways in reactive astrocytes, were differentially upregulates as compared to nascent astrocytes determined by PFSnet subnetwork analysis of differentially expressed genes (DEGs)18. Collectively, IL1β induced human spinal cord reactive astrocytes may exert various endogenous neuroprotective effects as demonstrated by the upregulation of critical axonal growth genes and downregulation of axonal inhibitory genes.In our study, we used IL1β to induce reactivity in , while all the cells were vimentin+. 24 hours after exposure to 100 ng/ml of IL1β24; the astrocytes acquired bipolar shape and a shrunken morphology with extensive elongated processes  were shorter as compared to IL1β treated astrocytes  .We tested the homogeneity of the nascent human spinal cord astrocytes by staining with astrocyte markers: Glial Fibrillary Protein (GFAP) and vimentin Fig. 19–22. Prses Fig. . The avetes Fig. . This chch) Fig. .Figure IL6 (223 folds), CXCL5 (205 folds), and C15orf48  (108 folds) were upregulated. EPHA7 was the most downregulated gene, followed by CEMIP and MTUS1 by −20, −18 and −18 folds, respectively. The complete list of changes in genes expression is provided in Supp. File + or GFAP- astrocytes were the main contributory factor in these transcriptome changes, the fraction of GFAP+ cells were first determined in control and in reactive astrocytes. The percentage of GFAP+ cells in reactive astrocytes was increased from 69.0 ± 5% in control to 94.6 ± 0% (p = 0.00152)  (p = 3.4 × 10−6). Likewise, CXCL5 + 6+ population fraction increased by more than 11 folds in reactive astrocytes from control . To determine the distribution of these IL6+ and CXCL5+ 6+ cells among GFAP+/− reactive astrocytes, the fraction of IL6+/GFAP+ and CXCL5+ 6+/GFAP+ reactive astrocytes were tabulated. 94.9 ± 1% GFAP+ reactive astrocytes were also positive with IL6+, while 66.7 ± 4% of GFAP+ reactive astrocytes were also stained with CXCL5+ 6 , CXCL6 (p = 2.34 × 10−6) and NMES1 (p = 6.4 × 10−7). The fold changes were respectively (IL6) 159.8 ± 37, (CXCL6) 53.0 ± 9 and (NMES1)133.1 ± 15 folds from control  is the most upregulated (3.24 folds), while matrilin2 (MATN2) (−2.37 folds) is the most downregulated axonal permissive genes. On the other hand, Slit Guidance Ligand (SLIT2) (2.54 folds) and Dorsal Inhibitory Axon Guidance Protein (DRAXIN) (2.52 folds) were the most upregulated genes involves in axonal growth inhibitory molecules. 8 out of 13 genes regulating axonal growth inhibitory molecules were down regulated, with Roundabout Guidance Receptor 2 (ROBO2) being the most downregulated genes in reactive astrocytes (−2.5 folds). Additionally, various matrix metallopeptidase and hyaluronan synthases were upregulated , PFSnet analysis revealed that actin cytoskeleton signaling pathway is one of the most altered pathway. As numerous pathways were differentially regulated in reactive astrocyte, our aim was to focus on main pathways that have critical role in axonal growth and development. We found that reactive astrocytes affect two subnetworks involved in axonal attraction and repulsion; FYN & RASGAP (Ras GTPase-activating protein 1) and SFK (Src family tyrosine kinase). In the “Axonal Guidance Signaling”, the axonal attraction and outgrowth seems to be regulated mainly by FYN and RASGAP were then analyzed with KEGG human pathways database to reveal affected biological pathways in human spinal cord reactive astrocytes. Using subnetwork analysis (PFSnet) within each pathway, it is revealed that reactive astrocytes differentially regulates subnetworks of various pathways, as compared to nascent astrocytes Fig. –446. Consis Figs –2, PFSnIn this study, we report the genome wide transcriptome profile of IL1β induced human spinal cord reactive astrocytes. We have studied a comprehensive subnetwork analysis based transcriptome pattern, which reveals the genes and their associated molecular function along with the important signaling pathways that regulate reactive astrogliosis. This provides a molecular roadmap to facilitate intervention and to optimize the benefits of reactive astrogliosis by either augmenting subnetworks of axonal attraction in the “Axonal Guidance Signaling” pathway or inhibiting signaling cascades in the “Axonal Repulsion” subnetwork.CXCL5 and NMES1 are among the most upregulated genes by 205 and 108 folds respectively. The association of NMES1 with human spinal cord reactive astrocytes is not well-known currently. We speculate that a very likely role of C15orf48 or commonly known as NMES1 may be in the regulation of the cell-cycle process, as its downregulation has been associated with potential tumorigenesis. Similarly, rat model of reactive-astrocyte using lipopolysaccharide  and middle carotid arterial occlusion  to induce injury did not identify CXCL5 expression in their clusters of differentially expressed genes. Hence, we deduce that reactive astrocytes are a heterogeneous cell population whose molecular identity is based on the type of injury and origin of host cells49.50, leading to morphology changes in reactive astrocytes14. The reorganization of the actin cytoskeleton is affected by inflammatory cytokines and is closely related to the “Focal Adhesion” and “ECM Receptor Interaction” pathways. A component of MAPK signaling pathway, ERK phosphorylation has been shown to be increased after SCI51. However, our PFSnet subnetwork analysis suggest that for the “MAPK signaling pathway”, the “JNK and p38 MAPK” sub-networks is activated in reactive astrocytes, while the classical “MAPK kinase sub-pathway” is down regulated. This selective subnetwork activation has not been clearly defined previously in reactive astrogliosis. Although the TGF-β signaling pathway is known to be involved in astrocyte reactivity, its exact signaling cascade has not been well-established in reactive astrocytes. Our findings indicate that one subnetwork of the pathway, involving ERK is upregulated while the other subnetwork involving the RhoA and PP2A is down regulated. Subnetwork analysis offers the advantage of differential view within a biological pathway, rather than revealing only an overall general alteration of biological pathway.The changes in the “Actin Cytoskeleton Signaling” pathway can be attributed to the change of the cytoskeleton into the more diffuse and ring-structure actin filaments(BDNF) in human spinal cord reactive astrocytes. The other neurotrophic factor that was upregulated was nerve growth factor (NGF). These two factors are critical in development, survival and regeneration of axons. Our findings thus suggest that early onset of reactive astrocytes could potentially promote neuronal and axonal development. Reactive astrocytes in the glial scar thus have the potential to secrete multiple axon-growth-permissive molecules, thus improving the microenvironment at and around the epicenter of injury. This will facilitate neuronal repair and regeneration. Recent findings also suggest that BDNF regulates the development of oligodendrocyte precursor cells56. Our results suggest that the primary axonal attraction aspect of reactive astrocytes, could be attributed to RasGAP and FYN over expression. Additionally, we also report the upregulated of “VEGF signaling” pathway, which has neurotrophic and neuroprotective effects on neuronal cells57.One intriguing observation was an increase in neurotrophic factors and “Axonal Guidance Signaling” pathway, particularly brain derived neurotrophic factor We also identified that there was an absence of up regulation of CSPGs genes. It has been known that CSPGs are major axonal growth inhibitor in glial scar. Our results present the possibility that reactive astrocytes may not be the main contributor of CPSGs at the epicenter and around the injury. Furthermore, we also reported the upregulation of hyaluronan synthases, which are enzymes for producing hyaluronan. The overexpression of hyaluronan is known to improve SCI recovery by reducing the lesion, and pro-inflammatory cytokines suggesting another neuroprotective properties of reactive astrocytes.MMP3, MMP1, and MMP12 belonging to the cluster of matrix proteins were found to be upregulated in reactive astrocytes. These genes have been associated with recruitment and migration of nascent astrocytes to the site of reactive astrocyte. Furthermore, MMP3 and MMP12 overexpression has been associated with remyelination. However, an increase in expression of matrix metalloproteases is known to enhance brain blood barrier (BBB) permeability and immune cells infiltration, resulting in inflammation post-CNS injury58. Our results indicate the upregulation of collagen and tenascin as well as downregulation of laminin and fibronectin in the ECM-receptor interaction pathways. In particular, collagen is critical in the early phase of tissue repair59 and the role of tenascin, in promoting neural outgrowth is controversial. Laminin and fibronectin are well-known for promoting neurite outgrowth. Collectively, we interpret that the ECM changes in reactive astrocytes may potentially induce both neuro-permissive and inhibitory effects to modulate axonal regeneration and growth66.Modification of extracellular matrix is an important element in formation of glial scar and modulation of axonal growth, regeneration and neuronal development post-CNS injury. 67. This include higher expression of pro-inflammatory miRNAs expression in adult astrocytes as compared to fetal68, while lower expression of fetal germinal matrix miRNAs in adult astrocytes as compared to fetal67. The higher expression of matrix associated miRNA could reflect the migrating status of the developing astrocytes in fetal CNS. Furthermore, young astrocytes also expressed genes involved in “neuronal differentiation”69. As our study is based on reactive astrogliosis in fetal astrocytes, it remains to be explored if neuroprotective and axonal regeneration potentials can replicated in adult reactive astrocytes. Also, our analysis indicates that both neuroprotective and inhibitory genes are activated in reactive astrocytes post 24 hours of IL1β exposure, suggesting that a balance of these gene expressions may regulate the functionality of reactive astrocytes.In our report, we presented a fetal human spinal cord reactive astrogliosis induced by IL1β. Although the genomic profiles of fetal astrocytes and adult astrocytes are comparable, notable differences in expression has been identified from previous literature3. The cells were cultured in Astrocytes Basal Medium (ScienCellTM) supplemented with 2% FBS (ScienCellTM), 1% Astrocyte growth supplement (ScienCellTM) and 1% of penicillin-streptomycin (ScienCellTM). The human astrocytes cells were plated on Matrigel (Corning) coated polystyrene 35mm plates with cell density of 1 × 105 cells. 24 hours after plating, cells were washed with PBS (HyClone™). Fresh medium containing IL1β (InvitrogenTM) at 100 ng/ml concentration was added and incubated for 24 hours in 5% CO2 at 37 °C.Human astrocytes derived from the spinal cord (19 weeks old fetus) was purchased from ScienCell Research Laboratories (Cat. No: 1820)In vitro: Human spinal cord astrocytes were grown on four wells matrigel coated dishes (Ibidi Inc.) at density of 2 × 104. They were fixed with 4% paraformaldehyde (PFA) in PBS for 10 minutes at 4 °C. The fixative solution was removed and cells were washed three times with PBS for 5 min each at 4 °C. Cells were permeabilized and blocked with blocking buffer for 30 minutes at 4 °C. Primary antibodies  [Millipore],  [sc-1265 Santa Cruz],   and  [RD system] was diluted in blocking buffer and incubated overnight at 4 °C. Samples were then washed three times with washing buffer for 10 minutes each at 4 °C. Secondary antibodies were added  in blocking buffer and cells were incubated at room for 4 hours. Cells were then washed three times with washing buffer for 5 minutes each at room temperature. Nuclei were stained with DAPI for 10 min at room temperature. DAPI solution was aspirated and washed once washing buffer for 5 minutes at room temperature was performed. Mounting medium DAKO was added and the samples were incubated at 4 °C overnight.(i) Cells were collected after exposure to IL1β for 24 hours, Accutase (StemPro®) was added to detach the cells. RNA was extracted using RNeasy Mini Kit  per manufacturer specifications.(i) All RNA samples were quality checked prior to microarray analysis in accordance to Affymetrix recommended protocols. Affymetrix 3′ IVT PLUS reagent kit was used. All RNA samples were assessed with spectrophotometric measurements  and quality RNA Integrity Number (RIN) with Agilent Bioanalyzer, (ii) for target preparation, 100 ng of total RNA was reverse transcribed to generate cDNA, and later used as template to generate biotin-labeled amplified RNA (aRNA). aRNA was then fragmented and hybridized to Affymetrix Human U133 Plus 2.0 Arrays, for 16 hours . Affymetrix Human U133 Plus 2.0 Arrays (HG-U133 Plus 2.0) comprised of 1,300,000 unique oligonucleotides, encompassing 47, 000 transcripts and variants, representing approximately 39, 000 of well characterized human genes, providing a complete coverage to the human genes. All samples were processed with similar reagent kit, were washed, stained and scanned with Affymetrix 300 7 G scanner. Scanned images were assessed for hybridization efficiency. (iii) Prior to microarray analysis, quality control checks were also carried out on the array. The 3′/5′ ratio of housekeeping genes, presences of spike controls, background value, raw Q noise, scaling factor, and percent of genes presence were evaluated. Signal intensity ratio of 3′/5′ probe sets is important in establishing good cDNA synthesis, integrity of starting RNA and hybridization properties. In order to obtain reliable and accurate data comparison, the background intensities were measured and were made sure to be consistent and closed to each other. Additionally, the raw Q noise was also measured by performing pixel to pixel variations in background intensities. This is to make sure that the variations in the digitized signal observed by the scanners as it samples the probe array’s surface is consistent and close to each other. The measure of brightness of the array which may vary between arrays to array was also normalized to a standard level. This normalization will be important in comparing array to array data. To assess the hybridization, washing and staining steps quality, bacteria spike controls were performed. These are probe sets hybridized by pre-labeled bacterial spike controls . Finally, Poly A control was also carried out to identify problems with target preparation. These are a set of poly-adenylated RNA spikes of Lys, Phe, Thr, and Dap, prepared in staggered concentration. These are prepared together with the samples throughout cDNA synthesis onwards.(Supp. Table (Supp. Table ). The array was also quality checked with consistent background intensity ranging from 30.781324 to 40.71225 (Supp. Table ). The normalized scaling factors was done to standardize the measure of brightness of the array, and the result of the array falls within the acceptable range of below 3-fold (Supp. Table ). In the bacteria spike controls, all samples also showed correct signal intensities of the control including BioB, suggesting good hybridization  ratio for 260/280 m, (>1.8) and 260/230 (>2.0) p < 0.05).For the changes in differential genes expression level, Affymetrix Transcriptome Console was used to analyze the level of expression for each individual gene. One-way between subject ANOVA, was used to assess the statistical significance , GFAP , and Cxcl 6  NMES1  were detected with the fluorescent double-stranded DNA binding dye, SYBR Green. qRT-PCR amplification was performed in triplicates for each sample and the results were replicated in four independent experiments. Gel electrophoresis and melting curve analyses were performed to validate PCR product sizes. The expression level of each gene was normalized against β-actin using the comparative CT method70.qRT-PCR was performed on ViiA™ 7 Real-Time PCR System (Applied Biosystems™) using SYBR Green PCR Master Mix reagent (Applied Biosystems ™). Specific PCR products for t-distribution is used as the null distribution for estimating the statistical significance of subnetworks scored in PFSNet. An additional criteria set was that any subnetwork tested for statistical significance, has to be highly expressed in all the samples in the corresponding group (control/IL1β). Changed in subnetworks were analyzed with pathway information from the PathwayAPI database34 which contains the aggregation of human pathways from KEGG71 and Ingenuity. Expression data is preprocessed using the espresso function in R affy package336. Pathways maps were generated based on KEGG pathways database tool3. Each subnetwork was ranked by their effect size. Effect size is a quantitative measure of difference between two groups. In our study, the effect size for each subnetwork was computed as the standardized mean of paired difference “(mean of paired differences)/(standard deviation of paired differences)” between PFSNet scores, corresponding to the control and reactive astrocyte groups73. As the sample size is small, multiple-testing correction was not performed since the p-value of the same gene fluctuates in a small range, with a large portion in the insignificant part even though the gene is differentially expressed by construction. Multiple-testing correction approaches would simply shift the null-hypothesis rejection threshold left-wards and thus would be insensitive against the wide fluctuation range in the p-value of this gene. We have also discuss similar approach previously63.Candidate subnetworks were generated by inducing connected components on known biological pathways with highly expressed genes in each phenotype. Two scores are computed for each subnetwork; these scores denote the level of expression of the subnetwork in majority of the samples in each phenotype. Finally, the difference of the two scores is tested for statistical significance. The theoretical 2 square visualized with (Plan-APOCHROMAT Zeiss NA = 0.45) 10X objective lens. 3 slices with 400 um between each of them were used for each rat. For in vitro cell length and processes quantification, 20X Plan-APOCHROMAT (NA = 0.8) objective lens was used. Cells that displayed processes were selected for quantification since not all the cells displayed a morphology that included processes. The main primary process of the cell was defined as any extension protruding from the cell body. The length of the primary process was defined as the distance from the center of the nuclei to the tip of the extended process. Only the main primary processes were used for quantification since very few cells displayed branching processes after exposure to IL-1B. For morphological quantification, the grayscale images were converted to binary type using ImageJ and their threshold was adjusted to clearly demarcate the cell boundary. The wand tool was used to outline the cellular perimeter in order to measure the cell surface area. Measuring was done using the line tool in Image J. Imaging settings were fixed for all groups within each experiments.The samples were imaged using ZEISS LSM 810 confocal microscopy instrument. Image J (Fiji) was used for image analysis and quantification. Tile scan and stitching were performed with ZEN blue software to image the entire spinal cord slice. Quantification for spinal cord GFAP intensity was done over 5 random Region of Interest (ROIs), covering the grey and white matter. The ROI is a 200 × 200 μmt-test for significance .All results were expressed as mean ± SEM, unless stated. Experiments were repeated independently for four times (n = 4) unless stated. Statistical significance was evaluated with unpaired student Supplementary DataMicroarray array results