Expression of Glucose Transporters in the Prelaminar Region of the Optic-Nerve Head of the Pig as Determined by Immunolabeling and Tissue Culture

Background: To develop the use of cultured tissue of the prelaminar optic nerve of the pig to explore possible alterations of the astrocyte-axon metabolic pathways in glaucoma, we map the distribution of the glucose transporters GLUT1 and GLUT3 in fresh and cultured tissue.Methods: We monitor cell survival in cultures of the prelaminar optic-nerve tissue, measuring necrosis and apoptosis markers biochemically as well as morphologically, and establish the presence of the glucose transporters GLUT1 and GLUT3. We map the distribution of these transporters with immunolabeling in histological sections of the optic nerve using confocal and electronic transmission microscopy.Results: We find that the main death type in prelaminar culture is apoptosis. Caspase 7 staining reveals an increment in apoptosis from day 1 to day 4 and a reduction from day 4 to day 8. Western blotting for GLUT1 shows stability with increased culture time. CLSM micrographs locate GLUT1 in the columnar astrocytes and in the area of axonal bundles. Anti-GLUT3 predominantly labels axonal bundles. TEM immunolabeling with colloidal gold displays a very specific distribution of GLUT-1 in the membranes of vascular endothelial cells and in periaxonal astrocyte expansions. The GLUT-3 isoform is observed with TEM only in axons in the axonal bundles.Conclusions: Tissue culture is suitable for apoptosis-induction experiments. The results suggest that glucose is transported to the axonal cleft intracytoplasmically and delivered to the cleft by GLUT1 transporters. As monocarboxylate transporters have been reported in the prelaminar region of the optic-nerve head, this area is likely to use both lactate and glucose as energy sources.This work was supported by a grant from the Consejería de Salud, Junta de Andalucia, Spain, Project PI-0655-2013

Measurement of the cosmic ray spectrum above 4×10184{\times}10^{18} eV using inclined events detected with the Pierre Auger Observatory

A measurement of the cosmic-ray spectrum for energies exceeding $4{\times}10^{18}$ eV is presented, which is based on the analysis of showers with zenith angles greater than $60^{\circ}$ detected with the Pierre Auger Observatory between 1 January 2004 and 31 December 2013. The measured spectrum confirms a flux suppression at the highest energies. Above $5.3{\times}10^{18}$ eV, the "ankle", the flux can be described by a power law $E^{-\gamma}$ with index $\gamma=2.70 \pm 0.02 \,\text{(stat)} \pm 0.1\,\text{(sys)}$ followed by a smooth suppression region. For the energy ($E_\text{s}$) at which the spectral flux has fallen to one-half of its extrapolated value in the absence of suppression, we find $E_\text{s}=(5.12\pm0.25\,\text{(stat)}^{+1.0}_{-1.2}\,\text{(sys)}){\times}10^{19}$ eV.Comment: Replaced with published version. Added journal reference and DO

Energy Estimation of Cosmic Rays with the Engineering Radio Array of the Pierre Auger Observatory

The Auger Engineering Radio Array (AERA) is part of the Pierre Auger Observatory and is used to detect the radio emission of cosmic-ray air showers. These observations are compared to the data of the surface detector stations of the Observatory, which provide well-calibrated information on the cosmic-ray energies and arrival directions. The response of the radio stations in the 30 to 80 MHz regime has been thoroughly calibrated to enable the reconstruction of the incoming electric field. For the latter, the energy deposit per area is determined from the radio pulses at each observer position and is interpolated using a two-dimensional function that takes into account signal asymmetries due to interference between the geomagnetic and charge-excess emission components. The spatial integral over the signal distribution gives a direct measurement of the energy transferred from the primary cosmic ray into radio emission in the AERA frequency range. We measure 15.8 MeV of radiation energy for a 1 EeV air shower arriving perpendicularly to the geomagnetic field. This radiation energy -- corrected for geometrical effects -- is used as a cosmic-ray energy estimator. Performing an absolute energy calibration against the surface-detector information, we observe that this radio-energy estimator scales quadratically with the cosmic-ray energy as expected for coherent emission. We find an energy resolution of the radio reconstruction of 22% for the data set and 17% for a high-quality subset containing only events with at least five radio stations with signal.Comment: Replaced with published version. Added journal reference and DO

Measurement of the Radiation Energy in the Radio Signal of Extensive Air Showers as a Universal Estimator of Cosmic-Ray Energy

We measure the energy emitted by extensive air showers in the form of radio emission in the frequency range from 30 to 80 MHz. Exploiting the accurate energy scale of the Pierre Auger Observatory, we obtain a radiation energy of 15.8 \pm 0.7 (stat) \pm 6.7 (sys) MeV for cosmic rays with an energy of 1 EeV arriving perpendicularly to a geomagnetic field of 0.24 G, scaling quadratically with the cosmic-ray energy. A comparison with predictions from state-of-the-art first-principle calculations shows agreement with our measurement. The radiation energy provides direct access to the calorimetric energy in the electromagnetic cascade of extensive air showers. Comparison with our result thus allows the direct calibration of any cosmic-ray radio detector against the well-established energy scale of the Pierre Auger Observatory.Comment: Replaced with published version. Added journal reference and DOI. Supplemental material in the ancillary file

Reducing the environmental impact of surgery on a global scale: systematic review and co-prioritization with healthcare workers in 132 countries

Abstract Background Healthcare cannot achieve net-zero carbon without addressing operating theatres. The aim of this study was to prioritize feasible interventions to reduce the environmental impact of operating theatres. Methods This study adopted a four-phase Delphi consensus co-prioritization methodology. In phase 1, a systematic review of published interventions and global consultation of perioperative healthcare professionals were used to longlist interventions. In phase 2, iterative thematic analysis consolidated comparable interventions into a shortlist. In phase 3, the shortlist was co-prioritized based on patient and clinician views on acceptability, feasibility, and safety. In phase 4, ranked lists of interventions were presented by their relevance to high-income countries and low–middle-income countries. Results In phase 1, 43 interventions were identified, which had low uptake in practice according to 3042 professionals globally. In phase 2, a shortlist of 15 intervention domains was generated. In phase 3, interventions were deemed acceptable for more than 90 per cent of patients except for reducing general anaesthesia (84 per cent) and re-sterilization of ‘single-use’ consumables (86 per cent). In phase 4, the top three shortlisted interventions for high-income countries were: introducing recycling; reducing use of anaesthetic gases; and appropriate clinical waste processing. In phase 4, the top three shortlisted interventions for low–middle-income countries were: introducing reusable surgical devices; reducing use of consumables; and reducing the use of general anaesthesia. Conclusion This is a step toward environmentally sustainable operating environments with actionable interventions applicable to both high– and low–middle–income countries

Schematic drawing of the experimental arrangement.

<p>The prelaminar region of the optic nerve head is excised and cultured in shallow medium to allow an adequate supply of oxygen to reach the tissue. As reported elsewhere, the axons are destroyed, leaving a matrix of astrocytes and capillaries that adapt to culture conditions. Microscopy (LM, TEM, and CLSM, including immunolabeling) is used to show that the tissue retains normal characteristics, including the important zonula adherens, and contains the glucose transport molecules GLUT1 and GLUT3. A comparison between the freshly excised and the cultured tissues by western blot shows the proportional quantities of the coupled molecules under study. This approach was followed in a previous report for MCTs and was performed here for GLUTs. (LM: light microscopy; TEM: transmission electron microscopy; CLSM: confocal laser scanning microscopy).</p

A to H. Tissue culture of the prelaminar region, JC-1 staining.

<p>A sequence showing the transformation from normal mitochondria into vesicular, inflated mitochondria. Healthy mitochondria are small compared to the nucleus (blue fluorescence). The normal metabolic activity of the mitochondrial membrane concentrates the dye (JC-1) on the inside and favors the formation of J-aggregates that fluoresce in bright red (A). As the ionic activity of the membrane decreases, the mitochondrion swells, the dye is less concentrated, and the hue changes from red to orange and finally to green (<b>B</b> to <b>H</b>) before mitochondrial disruption. This effect indicates mitochondrial-activated apoptosis as a main path for cell loss in prelaminar tissue culture. Here, the inflation of the mitochondria results from the decay of ionic pump function, which manifests as an increase in mitochondrial size and dilution of the dye from bright red to orange, yellow and green when increasingly diluted.</p

Histology of the prelaminar explant LM and TEM.

<p><b>a</b>) Semithin section of a full-length prelaminar explant following excision. The thickness varies between 100 and 300 microns, being thinner in the center. The central thickness in this case is approximately 280 microns. Toluidine blue inclusion, epoxy resin; magnification in the figure. <b>b</b>) Central zone (green square) in a). The arrangement in columns of astrocytes is highlighted by the dotted line in part of the section. Axonal bundles pass between the astrocytic columns. The area of the orange pentagon includes a capillary as part of a column and a portion of an axonal bundle. This type of section allows for GLUT-1 labeling in the endothelial vascular cells to serve as a positive control for axonal labeling of GLUT-1. <b>c</b>) The area enclosed in the red square is formed exclusively by axons wrapped in an extremely tight web of astrocytic expansions. The complexity of the arrangement is reminiscent of the neuropil in the central nervous system. The TEM micrograph shows three axons (Ax) and numerous astrocytic expansions. This type of section allows for the identification of the involved cells even in the absence of membrane counterstaining, which is required for immunolabeling with gold micelles. Cell identification is aided by the presence of GFAP and Neurofilament staining. The magnification is indicated in the figures.</p