Assessing groundwater denitrification spatially is the key to targeted agricultural nitrogen regulation.

Globally, food production for an ever-growing population is a well-known threat to the environment due to losses of excess reactive nitrogen (N) from agriculture. Since the 1980s, many countries of the Global North, such as Denmark, have successfully combatted N pollution in the aquatic environment by regulation and introduction of national agricultural one-size-fits-all mitigation measures. Despite this success, further reduction of the N load is required to meet the EU water directives demands, and implementation of additional targeted N regulation of agriculture has scientifically and politically been found to be a way forward. In this paper, we present a comprehensive concept to make future targeted N regulation successful environmentally and economically. The concept focus is on how and where to establish detailed maps of the groundwater denitrification potential (N retention) in areas, such as Denmark, covered by Quaternary deposits. Quaternary deposits are abundant in many parts of the world, and often feature very complex geological and geochemical architectures. We show that this subsurface complexity results in large local differences in groundwater N retention. Prioritization of the most complex areas for implementation of the new concept can be a cost-efficient way to achieve lower N impact on the aquatic environment.

Managing the remediation strategy of contaminated megasites using field-scale calibration of geo-electrical imaging with chemical monitoring.

Groundwater contamination is a threat to drinking water resources and ecosystems. Remediation by injection of chemical reagents into the aquifer may be preferred to excavation to reduce cost and environmental footprint. Yet, successful remediation requires complete contact between contamination and reagents. Subsurface heterogeneities are often responsible for diffusion into low-permeable zones, which may inhibit this contact. Monitoring the spatial distribution of injected reagents over time is crucial to achieve complete interaction. Source zone contamination at megasites is particularly challenging to remediate and monitor due to the massive scale and mixture of contaminants. Source zone remediation at Kærgård Plantation megasite (Denmark) is monitored here, with a new methodology, using high-resolution cross-borehole electrical resistivity tomography (XB-ERT) imaging calibrated by chemical analyses on groundwater samples. At this site, high levels of toxic non-aqueous phase liquids (NAPL) are targeted by in-situ chemical oxidation using activated persulfate. It may take numerous injection points with extensive injection campaigns to distribute reagents, which requires an understanding of how reagent may transport within the aquifer. A geophysical (XB-ERT) monitoring network of unprecedented size was installed to identify untreated zones and help manage the remediation strategy. The combination of spatially continuous geophysical information with discrete but precise chemical information, allowed detailed monitoring of sulfate distribution, produced during persulfate activation. Untreated zones identified in the first remediation campaign were resolved in the second campaign. The monitoring allowed adjusting the number of injection screens and the injection strategy from one campaign to the next, which resulted in better persulfate distribution and contaminant degradation in the second campaign. Furthermore, geophysical transects repeated over the timespan of a remediation campaign allowed high-resolution time-lapse imaging of reagent transport, which could in the future improve the predictability of transport models, compared to only using on a-priori assumptions of the hydraulic conductivity field.

Importance of different factors for modeling nitrate transport and retention in a tile-drained agricultural catchment with distance-based generalized sensitivity analysis.

Modeling of nitrate transport and retention in agricultural land use areas provides useful information to support water quality assessment and management. The accuracy and precision of model simulations are highly dependent on model input factors for which the appropriate values are generally difficult to determine and from which various uncertainties are induced into the modeling procedure. In this study, we applied a Distance-based Generalized Sensitivity Analysis (DGSA) to a high-resolution (25 × 25 m) nitrate transport and retention model for a tile-drained agricultural catchment (4.4 km2) to investigate the extent to which model input factors affect the spatially distributed nitrate retention. The input factors included the nitrate leaching from the root zone, the partitioning of nitrate into tile drainage and groundwater flux, the groundwater flux out of the catchment, the hydrogeological properties, and the denitrification rates in groundwater. The DGSA results were examined in both spatially lumped and distributed perspective. We found that the partitioning of nitrate into tile drainage and groundwater flux was the most important factor for modeling nitrate retention while the hydrogeological properties were secondary but also important. Conversely, the nitrate leaching from the root zone and denitrification rates in groundwater were noninfluential. By increasing the resolution of the DGSA analysis from catchment to model pixel, we found that input factors noninfluential on catchment scale were influential on pixel scale in discrete areas, and, as a general take-home-message, input factors influential on nitrate retention in at least 25 % of the model pixels were sensitive on catchment scale as well. Improved understanding of sensitivity of modelling nitrate retention may help the modelers and water managers to decide which input factors to prioritize in the modelling and data collection to improve the accuracy and precision of the model responses.

N-Map: High-resolution groundwater N-retention mapping and modelling by integration of geophysical, geological, geochemical, and hydrological data.

A key aspect of protecting aquatic ecosystems from agricultural nitrogen (N) is to locate (i) farmlands where nitrate leaches from the bottom of the root zone and (ii) denitrifying zones in the aquifers where nitrate is removed before entering the surface water (N-retention). N-retention affects the choice of field mitigation measures to reduce delivered N to surface water. Farmland parcels associated with high N-retention gives the lowest impact of the targeted field measures and vice versa. In Denmark, a targeted N-regulation approach is currently implemented on small catchment scale (approx. 15 km2). Although this regulatory scale is much more detailed than what has been used previously, it is still so large that regulation for most individual fields will be either over- or under-regulated due to large spatial variation in the N-retention. The potential cost reduction for farmers is of up to 20-30% from detailed retention mapping at the field scale compared to the current small catchment scale. In this study, we present a mapping framework (N-Map) for differentiating farmland according to their N-retention, which can be used for improving the effectiveness of targeted N-regulation. The framework currently only includes N-retention in the groundwater. The framework benefits from the incorporation of innovative geophysics in hydrogeological and geochemical mapping and modelling. To capture and describe relevant uncertainties a large number of equally probable realizations are created through Multiple Point Statistical (MPS) methods. This allows relevant descriptions of uncertainties of parts of the model structure and includes other relevant uncertainty measures that affects the obtained N-retention. The output is data-driven high-resolution groundwater N-retention maps, to be used by the individual farmers to manage their cropping systems due to the given regulatory boundary conditions. The detailed mapping allows farmers to use this information in the farm planning in order to optimize the use of field measures to reduce delivered agricultural N to the surface water and thereby lower the costs of the field measures. From farmer interviews, however, it is clear that not all farms will have an economic gain from the detailed mapping as the mapping costs will exceed the potential economic gains for the farmers. The costs of N-Map is here estimated to 5-7 €/ha/year plus implementation costs at the farm. At the society level, the N-retention maps allow authorities to point out opportunities for a more targeted implementation of field measures to efficiently reduce the delivered N-load to surface waters.

Automatic stochastic 3D clay fraction model from tTEM survey and borehole data.

In most urbanized and agricultural areas of central Europe, the shallow underground is constituted of Quaternary deposits which are often the most extensively used layers (water pumping, shallow geothermic, material excavation). All these deposits are often complexly intertwined, leading to high spatial variability and high complexity. Geophysical data can be a fast and reliable source of information about the underground. Still, the integration of these data can be time-consuming, it lacks realistic interpolation in a full 3D space, and the final uncertainty is often not represented. In this study, we propose a new methodology to combine boreholes and geophysical data with uncertainty in an automatic framework. A spatially varying translator function that predicts the clay fraction from resistivity is inverted using boreholes description as control points. It is combined with a 3D stochastic interpolation framework based on a Multiple Points Statistics algorithm and Gaussian Random Function. This novel workflow allows incorporating robustly the data and their uncertainty and requires less user intervention than the already existing workflows. The methodology is illustrated for ground-based towed transient electromagnetic data (tTEM) and borehole data from the upper Aare valley, Switzerland. In this location, a 3D realistic high spatial resolution model of clay fraction was obtained over the whole valley. The very dense data set allowed to demonstrate the quality of the predicted values and their corresponding uncertainties using cross-validation.

Controlled Subretinal Injection Pressure Prevents Damage in Pigs.

INTRODUCTION: Administration of retinal gene and stem cell therapy in patients with retinal degenerative diseases is in many cases dependent on a subretinal approach. It has been indicated that manual subretinal injection is associated with outer retinal damage, which may be explained by a high flow rate in the injection cannula. In the present porcine study, we evaluated flow-related retinal damage after controlled subretinal injection at different flow rates. METHODS: The flow rate through a 41G cannula was estimated at different injection pressures (6-48 pounds per square inch [PSI]) in an in vitro setup. A linear correlation between the flow rate and injection pressure was found from 6 to 32 PSI. In full anesthesia, 12 pigs were vitrectomized and received a controlled subretinal injection of 300 µL balanced saline solution at injection pressures of 14, 24, and 32 PSI (four in each group). Prior to surgery and 2 and 4 weeks after surgery, the eyes were examined by multifocal electroretinogram (mfERG) and fundus photographs. At the end of follow-up, the eyes were enucleated for histology. RESULTS: The in vitro flow study determined that the flow in a 41G cannula shifts from laminar to turbulent at 32 PSI and that the manual injection flow is turbulent. In the porcine study, we showed a significant difference in retinal pigment epithelium (RPE) damage between the three pressure groups (p = 0.0096). There was no significant difference in damage to the outer retina (p = 0.1526), but the high-pressure group (32 PSI) had the most outer retinal damage. The middle-pressure group (24 PSI) showed minimum retinal damage. There was no significant change in the mfERG ratios during follow-up. DISCUSSION/CONCLUSION: This study indicates that an injection pressure at approximately 24 PSI might be safe for subretinal delivery. Retinal damage at low injection pressures may be explained by mechanical damage to the RPE due to prolonged needle time in the subretinal space, while retinal damage at high pressures can be related to high flow in the injection cannula. Controlled subretinal injection pressure of 24 PSI showed minimum mechanical- and flow-related damage to the porcine retina.

Systemic vascular air embolus following CT-guided transthoracic needle biopsy: a potentially fatal complication.

Following an uncomplicated CT-guided transthoracic biopsy, a patient becomes unconscious and subsequently dies despite immediate cardiac resuscitation. The patient felt well during the procedure but started complaining about dizziness and chest pain when he sat up. When he again was put in a supine position, cardiac arrest was noted. A CT scan performed when the symptoms initiated was afterwards rigorously reviewed by the team and revealed air located in the left ventricle, aorta and right coronary artery.We present a rare but potentially lethal complication following CT-guided transthoracic needle biopsy-systemic vascular air embolus. Knowledge and evidence about the complication are sparse because of low incidence and varying presentation. However, immediate initiation of treatment can save a life, and awareness of the complication is therefore crucial.

Patients Own Safety Incidents Reports to the Danish Patient Safety Database Possess a Unique but Underused Learning Potential in Patient Safety.

OBJECTIVE: The aim of the study was to analyze how patients' own reports of safety incidents to the Danish Patient Safety Database can contribute to patient safety. BACKGROUND: Patient involvement enhances patient safety; however, there is a shortage of tools capable of systematically capturing and usage of patients' own reports. Since 2012, the Danish Patient Safety Database (DPSD) has comprised such a tool. METHODS: A total of 209,263 incident reports were compared across the following four reporting groups: patients, relatives, doctors, and nurses. Using thematic comparison, 300 narratives from each group were compared with respect to differences and similarities in the wording of the safety incident. RESULTS: Only a tiny proportion of safety incidents were reported by patients' themselves (1.4%). Most of these (86%) were accepted for processing in the DPSD. Almost 90% of the accepted incidents were classified successfully. Patients' own reports were longer, more often "less severe incidents," and more often reported by female patients. Thematic content analyses revealed incident descriptions from health professionals as terse, unemotional, and extensively using medical terminology and abbreviations. In contrast patients' reports were lengthy, emotional, and focused on relations to health personal, health consequences, and communication errors. CONCLUSIONS: Despite the very low ratio of patients reporting an observed incident to DPSD, the finding that most patients own reports are accepted and classified makes the DPSD a promising, comprehensive tool to capture patients' own reports. However, the rich, contextualized descriptions seem insufficiently captured by established nomenclature.

The Danish severe asthma register: an electronic platform for severe asthma management and research.

The evaluation and management of severe asthma patients require collection of comprehensive information, which is often a challenge in a busy outpatient clinic. The Danish Severe Asthma Register (DSAR) was designed as an electronic patient record form that captures operational clinical data and provides a clinical overview of the severe asthma patient. DSAR is a nationwide register; all patients in Denmark who are treated with biologics for severe asthma are included, and data are as a minimum entered at start of biological treatment, after four and 12 months of treatment, and hereafter annually. Currently, there are data from 621 treatment courses with biologics included in DSAR, with 71% of patients treated with anti-IL-5 drugs and 29% with an anti-IgE drug. Patients enter Patient Reported Outcome Measures electronically on tablets when they arrive in the outpatient clinic and their answers are immediately available to the clinician during the consultation. Nurses and doctors enter clinical data into DSAR during the consultation. DSAR offers immediate access to well-presented longitudinal overview and automatically creates a journal output that can be copy-pasted into the hospital's existing health record form. DSAR is also currently expanding with an app, to be used for monitoring of home-treatment. In addition to serving as an electronic patient record form, DSAR will also provide opportunities to monitor the real-life efficacy of biological treatment for severe asthma in Denmark, and it will be a valuable research platform that will aid in answering important research questions on severe asthma in the future.

Peptide Inhibitors of the α-Cobratoxin-Nicotinic Acetylcholine Receptor Interaction.

Venomous snakebites cause >100 000 deaths every year, in many cases via potent depression of human neuromuscular signaling by snake α-neurotoxins. Emergency therapy still relies on antibody-based antivenom, hampered by poor access, frequent adverse reactions, and cumbersome production/purification. Combining high-throughput discovery and subsequent structure-function characterization, we present simple peptides that bind α-cobratoxin (α-Cbtx) and prevent its inhibition of nicotinic acetylcholine receptors (nAChRs) as a lead for the development of alternative antivenoms. Candidate peptides were identified by phage display and deep sequencing, and hits were characterized by electrophysiological recordings, leading to an 8-mer peptide that prevented α-Cbtx inhibition of nAChRs. We also solved the peptide:α-Cbtx cocrystal structure, revealing that the peptide, although of unique primary sequence, binds to α-Cbtx by mimicking structural features of the nAChR binding pocket. This demonstrates the potential of small peptides to neutralize lethal snake toxins in vitro, establishing a potential route to simple, synthetic, low-cost antivenoms.

Neurotrophic Effects of Vascular Endothelial Growth Factor B and Novel Mimetic Peptides on Neurons from the Central Nervous System.

Vascular endothelial growth factor B (VEGFB) is a pleiotropic trophic factor, which in contrast to the closely related VEGFA is known to have a limited effect on angiogenesis. VEGFB improves survival in various tissues including the nervous system, where the effect was observed mainly for peripheral neurons. The neurotrophic effect of VEGFB on central nervous system neurons has been less investigated. Here we demonstrated that VEGFB promotes neurite outgrowth from primary cerebellar granule, hippocampal, and retinal neurons in vitro. VEGFB protected hippocampal and retinal neurons from both oxidative stress and glutamate-induced neuronal death. The VEGF receptor 1 (VEGFR1) is required for VEGFB-induced neurotrophic and neuroprotective effects. Using a structure-based approach, we designed short peptides, termed Vefin1-7, mimicking the binding interface of VEGFB to VEGFR1. Vefins were analyzed for their secondary structure and binding to VEGF receptors and compared with previously described peptides derived from VEGFA, another ligand of VEGFR1. We show that Vefins have neurotrophic and neuroprotective effects on primary hippocampal, cerebellar granule, and retinal neurons in vitro with potencies comparable to VEGFB. Similar to VEGFB, Vefins were not mitogenic for MCF-7 cancer cells. Furthermore, one of the peptides, Vefin7, even dose-dependently inhibited the proliferation of MCF-7 cells in vitro. Unraveling the neurotrophic and neuroprotective potentials of VEGFB, the only nonangiogenic factor of the VEGF family, is promising for the development of neuroprotective peptide-based therapies.

Loss of retinal tension and permanent decrease in retinal function: a new porcine model of rhegmatogenous retinal detachment.

PURPOSE: Permanent loss of visual function after rhegmatogenous retinal detachment can occur despite successful surgical reattachment in humans. New treatment modalities could be explored in a detachment model with loss of retinal function. In previous porcine models, retinal function has returned after reattachment, regardless of height and duration of detachment. Difference in retinal tension between the models and the disease might explain these different outcomes. This study investigates, for the first time in an in vivo porcine model, another characteristic of rhegmatogenous retinal detachment - the loss of retinal tension. METHODS: Left eyes (n = 12) of 3-month-old domestic pigs were included. Baseline multifocal electroretinogram (mfERG) and a fundus photograph were obtained following anaesthesia (isoflurane). The pigs were vitrectomized, saline was injected subretinally, and the RPE was removed. The eyes were evaluated at 2, 4 and 6 weeks after surgery. Four eyes were enucleated at each evaluation for histologic examinations. RESULTS: A retinal detachment structurally resembling rhegmatogenous retinal detachment was induced in 11 out of 12 pigs. MfERG amplitudes were significantly decreased despite partial reattachment four and 6 weeks after detachment. The retinal thickness decreased with 27%, the inner nuclear layer degenerated, Müller cells hypertrophied, and outer segments were lost. In the ganglion cell layer, cellularity increased and there was cytoplasmic staining with Cyclin D1. Vimentin and GFAP staining for glial cells increased. After 2 weeks of detachment, the ganglion cells had lost their nucleus and nucleolus. CONCLUSIONS: Loss of retinal tension in the detached retina seems to induce permanent damage with loss of retinal function. Death of ganglion cells, observed as soon as 2 weeks after detachment, explains the permanent loss of retinal function. The new model enables investigations of time-relationship between retinal detachment and lasting damage in addition to exploration of novel treatment modalities.

3D characterization of the subsurface redox architecture in complex geological settings.

Nitrogen (N) leaching caused by agricultural activities is one of the major threats to the aquatic ecosystems and public health. Moving from the agricultural soils through the subsurface and reemerging to the surface water, N undergoes various biogeochemical reactions along pathways in the subsurface, which occur heterogeneously in space and time. Thus to improve our understanding on the fate and distribution of N in the aquatic environment, detailed knowledge about the subsurface hydrogeological and biogeochemical conditions, especially the redox conditions, are essential. In this study, 3D information of the redox conditions termed the redox architecture was investigated in two Danish catchments with intensive agriculture underlain by glacial deposits. Towed transient electromagnetic (tTEM) resistivity was interpreted which reveals the subsurface geological structures at a few hectare scale. These geophysical data were integrated with sediment and water chemistry for the redox architecture interpretations. The top soils of both catchments are characterized as clay-till, but the tTEM showed that the subsurface hydrogeological structures are distinctively different. We identified three types of redox architectures in the studied catchments: 1) a planar redox architecture with a single redox interface; 2) a geological-window redox architecture with local complexity; and 3) a glaciotectonic-thrusted redox architecture with high complexity. The baseflow N load at the catchment outlets reflect the contributions of N via oxic pathways through the complex redox architectures of the subsurface. We conclude that in some landscapes, the redox architecture cannot be simplified as a single interface that roughly follows the terrain; hence, thorough investigations of the structural heterogeneity of the local redox architectures will be necessary to improve simulations of N evolution along pathways and quantifications of N attenuation under various mitigation scenarios.

Cross-borehole tomography with full-decay spectral time-domain induced polarization for mapping of potential contaminant flow-paths.

Soil contamination from industrial activities is a large problem in urban areas worldwide. Understanding the spreading of contamination to underlying aquifers is crucial to make adequate risk assessments and for designing remediation actions. A large part of the northern hemisphere has quaternary deposits consisting of glacial clayey till. The till often has a complex hydrogeological structure consisting of networks of fractures, sand stringers and sand lenses that each contribute to a transport network for water, free phase and dissolved contaminants. Thus, to determine the possible flow-paths of contaminants, the geology must be described in great detail. Normally, multiple boreholes would be drilled in order to describe the geology, but boreholes alone do not provide the needed resolution to map such sand lenses and their connectivity. Cross-borehole full-decay time-domain induced polarization (TDIP) is a new tool that allows for quantitatively mapping not only contrasts in bulk resistivity, but also contrasts in spectral IP parameters. We present a feasibility study with synthetic tests and a field application on a clayey moraine environment with embedded sand lenses, with hitherto unseen ground-truth verification. Indeed, the investigated area was above the water table, which allowed for digging out the entire area after the investigation for an unprecedented description of the lens interconnectivity. The TDIP data were acquired with a full-waveform acquisition at high sampling rate, signal-processed by harmonic denoising, background removal, and de-spiking, and subsequently the full-waveform data were stacked in log-increasing tapered gates (with 7 gates per decade). The resulting TDIP decays, with usable time-gates as early as two milliseconds, were inverted in terms of a re-parameterization of the Cole-Cole model. The inverted models of the field data show a remarkable delineation of the sand lenses/layers at the site, with structure in both the resistivity and the IP parameters matching the results from the ground-truthing. The synthetic examples show that in models both below and above the groundwater table, sand-lenses with thicknesses comparable to the vertical electrode spacing can be well resolved. This suggests that full-decay cross-borehole TDIP is an ideal tool for high-resolution sand-lens imaging.

Bruch's membrane allows unhindered passage of up to 2 µm latex beads in an in vivo porcine model.

PURPOSE: It has been proposed that changes in the permeability of Bruch's membrane play a role in the pathogenesis of age-related macular degeneration (AMD). This paper investigates, in an in vivo porcine model, the migration of fluorescent latex beads across the Bruch's membrane after subretinal injection. METHODS: Forty-one healthy eyes of 33 three-month-old domestic pigs received a subretinal injection of 0.5, 1.0, 2.0, or 4.0 µm fluorescent latex beads. Between three hours and five weeks after injection evaluations were performed with fundus photographs and histology. Fluorescent beads were identified in unstained histologic sections using the rhodamine filter with the light microscope. RESULTS: The fluorescent latex beads relocated from the subretinal space. Intact beads up to 2.0 µm were found in the choroid, sclera, and extrascleral space. The smaller beads were also found inside choroidal and extrascleral blood vessels. In contrast, the larger beads of 4.0 µm did not pass the Bruch's membrane. CONCLUSION: Subretinally implanted beads up to 2.0 µm pass the Bruch's membrane intact and cross the blood-ocular barrier. The intact beads are found in the choroid, sclera and inside blood vessels. The results give reason to consider the role of subretinal clearance and passage of Bruch's membrane in the development of AMD.

Neuropeptide Y treatment induces retinal vasoconstriction and causes functional and histological retinal damage in a porcine ischaemia model.

PURPOSE: To investigate the effects of intravitreal neuropeptide Y (NPY) treatment following acute retinal ischaemia in an in vivo porcine model. In addition, we evaluated the vasoconstrictive potential of NPY on porcine retinal arteries ex vivo. METHODS: Twelve pigs underwent induced retinal ischaemia by elevated intraocular pressure clamping the ocular perfusion pressure at 5 mmHg for 2 hr followed by intravitreal injection of NPY or vehicle. After 4 weeks, retinas were evaluated functionally by standard and global-flash multifocal electroretinogram (mfERG) and histologically by thickness of retinal layers and number of ganglion cells. Additionally, the vasoconstrictive effects of NPY and its involved receptors were tested using wire myographs and NPY receptor antagonists on porcine retinal arteries. RESULTS: Intravitreal injection of NPY after induced ischaemia caused a significant reduction in the mean induced component (IC) amplitude ratio (treated/normal eye) compared to vehicle-treated eyes. This reduction was accompanied by histological damage, where NPY treatment reduced the mean thickness of inner retinal layers and number of ganglion cells. In retinal arteries, NPY-induced vasoconstriction to a plateau of approximately 65% of potassium-induced constriction. This effect appeared to be mediated via Y1 and Y2, but not Y5. CONCLUSION: In seeming contrast to previous in vitro studies, intravitreal NPY treatment caused functional and histological damage compared to vehicle after a retinal ischaemic insult. Furthermore, we showed for the first time that NPY induces Y1- and Y2- but not Y5-mediated vasoconstriction in retinal arteries. This constriction could explain the worsening in vivo effect induced by NPY treatment following an ischaemic insult and suggests that future studies on exploring the neuroprotective effects of NPY might focus on other receptors than Y1 and Y2.

Localization, distribution, and connectivity of neuropeptide Y in the human and porcine retinas-A comparative study.

Neuropeptide Y (NPY) is a peptide neurotransmitter abundantly expressed in the mammalian retina. Since its discovery, NPY has been studied in retinas of several species, but detailed characterization of morphology, cell-type, and connectivity has never been conducted in larger mammals including humans and pigs. As the pig due to size and cellular composition is a well-suited animal for retinal research, we chose to compare the endogenous NPY system of the human retina to that of pigs to support future research in this field. In the present study, using immunohistochemistry, confocal microscopy and 3D reconstructions, we found NPY to be expressed in GABAergic and calretinin-immunoreactive (-ir) amacrine cells of both species as well as parvalbumin-ir amacrine cells of humans. Furthermore, we identified at least two different types of medium- to wide-field NPY-ir amacrine cells. Finally, we detected likely synaptic appositions between the NPY-ir amacrine cells and melanopsin- and nonmelanopsin-ir ganglion cells, GABAergic and dopaminergic amacrine cells, rod bipolar cells, and horizontal cells, suggesting that NPY-ir cells play diverse roles in modulation of both image and non-image forming retinal signaling. These findings extend existing knowledge on NPY and NPY-expressing cells in the human and porcine retina showing a high degree of comparability. The extensive distribution and connectivity of NPY-ir cells described in the present study further highlights the potential importance of NPY signaling in retinal function.

Early Discontinuation of Montelukast Treatment; A Danish Nationwide Utilization Study.

Montelukast, a leukotriene receptor antagonist, was marketed in 1998 as an oral supplementary treatment to patients with mild to moderate asthma. The aim of this study was to describe the early discontinuation pattern among montelukast users in Denmark in the period of 1 March 1998 to 31 December 2016, and to identify demographic characteristics possibly associated with early discontinuation. This nationwide drug utilization study was based on data collected from three nationwide Danish registers. All montelukast users who redeemed at least one prescription in the study period were identified. Early discontinuation was defined as failing to fill a second prescription for montelukast within at least a year after the initial montelukast prescription. Among 135,271 included montelukast users, 47,480 (35%) discontinued the use of montelukast after a single redeemed prescription. The trend in early discontinuation increased throughout the years. The most predominant demographic risk factors for early discontinuation were prescription for only nasal topical anti-allergic treatment up to a year prior to montelukast initiation [adjusted odds ratio (OR) 2.25; 95% confidence interval (CI) 2.13-2.38], as well as suspected off-label use (adjusted OR 2.02; 95% CI 1.97-2.08). Several risk factors were associated with a decreased risk of early discontinuation; most pronounced was a prescription of inhaled corticosteroids within a year up to montelukast initiation [adjusted OR 0.47 (95% CI 0.46-0.49)]. Early discontinuation was more pronounced after patent expiry in 2012 [adjusted OR 1.42 (95% CI 1.38-1.45)]. In conclusion, we found that early montelukast discontinuation increased during the last 19 years. Appropriateness of the treatment indication as estimated by concomitant prescription of adequate inhalation therapy was associated with a low risk of early discontinuation. A more pronounced early discontinuation was observed after patent expiry in 2012, which could reflect a more liberal approach to montelukast prescription.

Increased endothelin-1-mediated vasoconstriction after organ culture in rat and pig ocular arteries can be suppressed with MEK/ERK1/2 inhibitors.

PURPOSE: Even though retinal vascular changes following ischaemia have been poorly understood, the upregulation of vasoconstrictive endothelin-1 (ET-1) receptors (ETA /ETB ) following global cerebral ischaemia has been described. The aim of this study was to investigate whether or not the MEK/ERK1/2 pathway is involved in the observed upregulation and whether specific MEK/ERK1/2 inhibitors U0126 and trametinib can prevent it. METHODS: The aim was also to localize ETA and ETB receptors using immunohistochemistry in both fresh rat ophthalmic arteries and after 24-hr organ culture and study the receptors functionally using myography. Pig retinal arteries also underwent 24-hr organ culture to validate similar responses across species and the retinal vasculature. RESULTS: Results showed that following organ culture there is a significant increase in ET-1-mediated vasoconstriction, in particular via the ETB receptor. Furthermore, immunohistochemistry revealed a clear increase in pERK in the smooth muscle cells of rat ophthalmic artery. U0126 and trametinib were successful in attenuating the functional vasoconstriction in both rat and pig, as well as restoring immunofluorescence of pERK to fresh levels and counteracting ETB expression in the smooth muscle cells of the rat ophthalmic artery. CONCLUSION: This is the first study to show that the MEK/ERK1/2 pathway in responsible for the increase in functional vasoconstriction via ET-1 receptor in rat ophthalmic and pig retinal arteries. Furthermore, this study is the first to suggest a way of inhibiting and preventing such an increase. With these results, we suggest a novel approach in retinal ischaemia therapy.