Macular pigment measurement in clinics: controlling the effect of the ageing media.

We report a series of experiments designed to ensure that Macular Pigment Optical Density (MPOD) measurements obtained with a clinical instrument are not influenced by lens yellowing and ocular media optical density. These effects were determined in six subjects using seven Lee Colour Temperature Correcting filters to simulate changes in the transmittance of the ocular media with age. Calculated simulated age matched the data linking age and optical density reported in the literature, and the MPOD was independent of simulated age. The instrument allows an estimation of MPOD to be made which is based only on a foveal (centre-only) measurement rather than, as is conventional, making a comparison between foveal and peripheral measurements. We assessed the performance of this facility by comparing the centre-only estimate of MPOD with that obtained from both central and peripheral measurements in 5616 eyes. The 95% limits of agreement for the two estimates was 0.13 OD units.

The dazzle reflex: electrophysiological signals from ocular muscles reveal strong binocular summation effects.

Under dark adapted or dim conditions the mammalian visual system is carefully programmed to respond rapidly to the sudden onset of bright lights. This response, called the dazzle reflex, is controlled from sub-cortical structures of the brain. It is known anecdotally that exposure to a bright light when dark adapted induces an instinctive closure of one eye to reduce the pain associated with dazzle. This binocular summation of the dazzle response has not previously been reported. The dazzle reflex can be measured in human subjects by recording the electrical activity from surface electrodes located near the muscles around the eye. In this paper we report an investigation of the apparent binocular summation of the dazzle reflex using this technique. The data reveal a clear difference between monocular and binocular stimulation, with the binocular response being much larger than the monocular response. Furthermore this monocular/binocular difference arises only if the stimulus duration is longer than approximately 1 s. These observations are interpreted in terms of the known physiology of blink mechanisms.

Acute locking in revision total knee arthroplasty due to disengagement of the locking screw.

Acute locking of the joint in a replaced knee joint is very rare. This report describes an acute locking episode of a revised modular total knee replacement, occurring more than 2 years after surgery. A disengaged screw from the modular femoral component had lodged in the joint at the inferior pole of the patella and required urgent arthroscopic removal. There was no subsequent failure of the stem-condylar junction, nor loosening of the femoral component.

Cell column chromatography: a new research tool to quantify cerebral cell volume changes following chemically-induced anoxia/re-oxygenation.

OBJECTIVE: The roles of individual types of cerebral cells in contributing to brain edema are undefined. The objective of this study was to determine the role of cerebral cell-column chromatography in quantifying cell volumes of individual cerebral cell lines, under chemically-induced anoxia/re-oxygenation (A/R). METHODS: Cerebral endothelial cells (4 experiments) or type II astrocytes (4 experiments) were cultured to confluence on microcarrier beads. A chromatographic cell-column of 1.5 cm height was filled with non-treated cell-covered beads. The column was perfused at 1 ml/min with a balanced perfusate for one hour (Baseline). The perfusate was then switched to that containing 5 mM thioglycolic acid for one hour (Anoxia). Then the column was perfused with the normal perfusate for another two hours (Re-oxygenation). The total free space in the column, reversely reflecting cell volumes, was determined by averaged transit time (TTa) of a non-permeable flow tracer blue dextran. Decreased TTa means that cells swell, and vice versa. RESULTS: TTa in endothelial cell columns increased with a peak at 60 minutes of re-oxygenation. TTa in astrocyte columns decreased with a nadir at 30 minutes of re-oxygenation. CONCLUSION: Cell column chromatography can be used to determine the cerebral cell volume changes following chemically-induced anoxia/re-oxygenation.

Ifenprodil treatment is associated with a down-regulation of brain aquaporin 4 following cardiac arrest in rats.

OBJECTIVES: Ifenprodil, a NMDA receptor polyamine site antagonist, reduces experimental cardiac arrest (CA)-elicited brain edema, which is associated with an up-regulation of aquaporin 4 (AQP4), a brain water-selective channel. However, the interacting roles of NMDA receptors and AQP4 in CA-elicited brain edema are unknown. The objective of this study was to test our hypothesis that ifenprodil treatment is associated with a down-regulation of brain AQP4. METHODS: Twenty-five rats were assigned to normal controls (group 1, n = 6) or subjected to eight minutes of asphyxial CA treated with placebo (group 2, n = 9) or ifenprodil (group 3, n = 10). Ifenprodil at 10 mg/kg or normal saline of equal volume was given intraperitoneally, 5 minutes before CA. The density of AQP4 protein and actin bands was scanned and expressed as the ratios of the optical density of AQP4 relative to that of actin. The ANOVA analysis was used to compare the group differences. RESULTS: The ratios of the optical density of AQP4 to that of actin were 0.88 +/- 0.06 in group 1, 1.11 +/- 0.08 in group 2 (p < 0.05 vs. group 1), and 0.78 +/- 0.04 in group 3 (p < 0.01 vs. group 2; NS vs. group 1). CONCLUSION: Ifenprodil given before CA is associated with a downregulation of brain AQP4 in rats.

Matrix metalloproteinases are not involved in early brain edema formation after cardiac arrest in rats.

INTRODUCTION: Resuscitation from cardiac arrest (CA) often results in a poor neurological outcome possibly due to an incomplete understanding of the pathophysiology of brain injury following CA-induced global cerebral ischemia. Brain edema is an important manifestation after CA and is associated with significant morbidity and mortality. The matrix metalloproteinases (MMPs) contribute to brain edema formation following focal cerebral ischemia. The objective of this study was to investigate the role of an MMP inhibitor, GM6001, in CA-elicited brain edema. METHODS: Eighteen rats were subjected to normothermic (37.5 +/- 0.5 degrees C) CA induced by eight minutes of asphyxiation and assigned to a CA-control group (CA), an alcohol-placebo group (CA + ETOH), or a GM6001-treated group (CA + GM6001). GM6001 in 100% alcohol or a vehicle was given i.v. before CA to achieve a whole blood concentration of 10 microM. Animals were resuscitated with CPR, ventilation and epinephrine. Brain edema was determined by brain wet-to-dry weight ratio at one hour after resuscitation. FINDINGS: Brain wet-to-dry weight ratio was 4.86 +/- 0.09 in CA, 4.76 +/- 0.12 in CA + ETOH (p = 0.30 vs. CA), and 4.72 +/- 0.03 in CA + GM6001 (p = 0.17 vs. CA and 0.42 vs. CA + ETOH). INTERPRETATION: MMPs are not involved in brain edema formation one hour following CA.

Redox regulation of endothelial barrier integrity.

Intestinal ischemia-reperfusion is associated with the generation of reactive oxygen metabolites as well as remote, oxidant-mediated lung injury. Oxidants elicit endothelial redox imbalance and loss of vascular integrity by disorganizing several junctional proteins that contribute to the maintenance and regulation of the endothelial barrier. To determine the specific effect of redox imbalance on pulmonary vascular barrier integrity, microvascular permeability was determined in lungs of animals subjected to chemically induced redox imbalance. The effect of redox imbalance on microvascular permeability and endothelial junctional integrity in cultured lung microvascular cells was also determined. Whole lung and cultured pulmonary endothelial cell permeability both increased significantly in response to chemical redox imbalance. Thiol depletion also resulted in decreased endothelial cadherin content and disruption of the endothelial barrier. These deleterious effects of intracellular redox imbalance were blocked by pretreatment with exogenous glutathione. The results of this study suggest that redox imbalance contributes to pulmonary microvascular dysfunction by altering the content and/or spatial distribution of endothelial junctional proteins.

An improved Na+-selective microelectrode for intracellular measurements in plant cells.

The high background K+ concentration in plant cells is a problem for intracellular measurements of Na+ using ion-selective microelectrodes. The discrimination between Na+ and K+ of the microelectrode ionophore molecule limits the usefulness of this technique. A new Na+-selective microelectrode with an ionophore incorporating a tetramethoxyethyl ester derivative of p-t-butyl calix[4]arene has been developed. Microelectrodes made with this new sensor have superior selectivity for Na+ over K+ resulting in a lower limit of detection when compared with microelectrodes made using a commercially available ionophore (ETH227). Both types of microelectrodes were insensitive to changes in ionic strength and physiological ranges of pH, but only the calixarene-based electrodes showed no protein interference. To test the suitability of the calixarene-based microelectrodes for measurements in plants, they were used to measure Na+ in epidermal cells in the zone 10-20 mm from the root apex of barley (Hordeum vulgare L.). Seedlings were grown in a nutrient solution containing 200 mM NaCl for 1-6 d. The range of intracellular Na+ activity (a(Na)) measured varied from < or =0.1 mM (limit of detection) to over 100 mM, and these values increased significantly with time. The membrane potential (E(m)) of these cells was variable, but the values became significantly more negative with time, although there was no significant correlation between E(m) and a(Na). These intracellular measurements could not be separated into distinct populations that might be representative of subcellular compartments.

Macular pigment and risk for age-related macular degeneration in subjects from a Northern European population.

PURPOSE: Age and advanced disease in the fellow eye are the two most important risk factors for age-related macular degeneration (AMD). In this study, the authors investigated the relationship between these variables and the optical density of macular pigment (MP) in a group of subjects from a northern European population. METHODS: The optical density of MP was measured psychophysically in 46 subjects ranging in age from 21 to 81 years with healthy maculae and in 9 healthy eyes known to be at high-risk of AMD because of advanced disease in the fellow eye. Each eye in the latter group was matched with a control eye on the basis of variables believed to be associated with the optical density of MP (iris color, gender, smoking habits, age, and lens density). RESULTS: There was an age-related decline in the optical density of macular pigment among volunteers with no ocular disease (right eye: r(2) = 0.29, P = 0.0006; left eye: r(2) = 0.29, P < 0.0001). Healthy eyes predisposed to AMD had significantly less MP than healthy eyes at no such risk (Wilcoxon's signed rank test: P = 0.015). CONCLUSIONS: The two most important risk factors for AMD are associated with a relative absence of MP. These findings are consistent with the hypothesis that supplemental lutein and zeaxanthin may delay, avert, or modify the course of this disease.

Polynitroxyl-albumin (PNA) plus tempol attenuate lung capillary leak elicited by prolonged intestinal ischemia and reperfusion(1).

Stable nitroxyl radicals (nitroxides) are potential antioxidant drugs, and we have previously reported that linking nitroxide to biological macromolecules can improve therapeutic activity in at least two ways. First, polynitroxylated compounds such as polynitroxyl human serum albumin (PNA) are a novel class of high molecular weight, extracellular antioxidants. Second, compounds such as PNA can prolong the half-life of free (unbound, low molecular weight) nitroxides such as 4-hydroxy-2,2,6, 6-tetramethylpiperidine-N-oxyl (Tempol) in vivo. Unlike PNA, Tempol can readily access the intracellular compartment. Thus PNA can act alone in the extracellular compartment, or in concert with Tempol, to provide additional antioxidant protection within cells. In this study, we compared the abilities of PNA, Tempol, and the combination of PNA + Tempol to prevent lung microvascular injury secondary to prolonged gut ischemia (I, 120 min) and reperfusion (R, 20 min) in the rat. Pulmonary capillary filtration coefficient (K(f,c)) and lung neutrophil retention (tissue myeloperoxidase activity, MPO) were measured in normal, isolated rat lungs perfused with blood harvested from I/R rats. Blood donor rats were treated with drug during ischemia. Gut I/R resulted in a marked increase in pulmonary capillary coefficient and lung MPO. PNA + Tempol, but not PNA alone or Tempol alone, at the doses used, prevented the development of lung leak. None of the treatments had an effect on lung neutrophil retention. Anti-inflammatory therapeutic activity appeared to correlate with blood Tempol level: in the presence of PNA, blood Tempol levels were maintained in the 50-100 microM range vs. essentially undetectable levels shortly after Tempol was administered alone. In this model of lung injury secondary to prolonged gut I/R, lung capillary leak was prevented when the membrane-permeable compound Tempol was maintained in its active, free radical state by PNA.

Macular pigment optical density measurement: a novel compact instrument.

A compact device to derive the optical density of human macular pigment (MP) using heterochromatic flicker photometry is described. The validity of the system is assessed by measuring the optical density spectra of MP in 12 healthy subjects and comparing this with well-established previously published values. The mean spectral absorbance characteristics of MP across subjects corresponds well with accepted values. As reported in other studies, our measurements show a wide variation of MP optical densities between individuals. In our technique within-subject variability is low; standard deviations are between 0.025 and 0.15 in most cases. The overall optical density of MP ranged from 0.08 to 0.84 with a mean of 0.496 and standard deviation of 0.257 at 460 nm. The stimulus size was 0.95 degrees. The unique feature of the technique is that it allows free viewing (not Maxwellian View) of the stimulus, it can be conducted easily and quickly and does not need frequent re-calibration.

Inflammatory mediators induce sequestration of VE-cadherin in cultured human endothelial cells.

The mechanisms through which inflammatory mediators modify endothelial junctional structure are not well understood. Endothelial cells exposed to 1 mM H2O2, 0.1 mM histamine or 4 mM EDTA displayed decreased amounts of VE-cadherin on the cell surface in a time-dependent manner. H2O2 and EDTA-treated cells showed a sustained reduction in surface VE-cadherin, but histamine (0.1 mM) decreased cell surface VE-cadherin only at 5 and 15 min, not at 30 and 60 min. Sequestering of VE-cadherin could also be visualized as a decrease in immunofluorescent labeling of endothelial junctions in fixed, non-extracted monolayers. However, junctional staining was observed in these cells after membrane extraction. This decreased surface expression of VE-cadherin was actin-filament, but not PKC/MAP kinase dependent. VE-cadherin binding to the cytoskeleton was decreased by EDTA, but was not diminished by histamine or H2O2. Therefore, by promoting sequestration of junctional cadherins, inflammatory mediators may decrease adhesive bonds between apposed endothelial cells and increase solute permeability.

Pathophysiology of ischaemia-reperfusion injury.

Reperfusion of ischaemic tissues is often associated with microvascular dysfunction that is manifested as impaired endothelium-dependent dilation in arterioles, enhanced fluid filtration and leukocyte plugging in capillaries, and the trafficking of leukocytes and plasma protein extravasation in postcapillary venules. Activated endothelial cells in all segments of the microcirculation produce more oxygen radicals, but less nitric oxide, in the initial period following reperfusion. The resulting imbalance between superoxide and nitric oxide in endothelial cells leads to the production and release of inflammatory mediators (e.g. platelet-activating factor, tumour necrosis factor) and enhances the biosynthesis of adhesion molecules that mediate leukocyte-endothelial cell adhesion. Some of the known risk factors for cardiovascular disease (hypercholesterolaemia, hypertension, and diabetes) appear to exaggerate many of the microvascular alterations elicited by ischaemia and reperfusion (I/R). The inflammatory mediators released as a consequence of reperfusion also appear to activate endothelial cells in remote organs that are not exposed to the initial ischaemic insult. This distant response to I/R can result in leukocyte-dependent microvascular injury that is characteristic of the multiple organ dysfunction syndrome. Adaptational responses to I/R injury have been demonstrated that allow for protection of briefly ischaemic tissues against the harmful effects of subsequent, prolonged ischaemia, a phenomenon called ischaemic preconditioning. There are two temporally and mechanistically distinct types of protection afforded by this adaptational response, i.e. acute and delayed preconditioning. The factors (e.g. protein kinase C activation) that initiate the acute and delayed preconditioning responses appear to be similar; however the protective effects of acute preconditioning are protein synthesis-independent, while the effects of delayed preconditioning require protein synthesis. The published literature in this field of investigation suggests that there are several potential targets for therapeutic intervention against I/R-induced microvascular injury.

Effect of femoral fracture and intramedullary fixation on lung capillary leak.

BACKGROUND: Pulmonary injury is an important complication in the trauma patient with long-bone fractures. The purpose of this study was to determine the effect of femoral fracture or fracture and intramedullary fixation on lung capillary leak. The contribution of leukocytes to lung injury in this model was also determined. METHODS: The pulmonary capillary filtration coefficient was determined in lungs of rats after femur fracture or fracture and reamed or unreamed intramedullary fixation. Pulmonary arterial vascular resistance and lung neutrophil content were also determined. RESULTS: Fracture alone did not cause lung injury, whereas fracture and intramedullary fixation elicited lung capillary leak. Fracture alone and intramedullary fixation increased pulmonary vascular resistance, whereas unreamed intramedullary fixation caused lung leukosequestration. CONCLUSION: Femoral fracture alone does not cause an increase in pulmonary microvascular permeability. Femoral fracture and intramedullary fixation causes lung capillary leak, which is not increased by reaming the femoral canal.

Aspiration of activated charcoal elicits an increase in lung microvascular permeability.

BACKGROUND: Gastric decontamination with orally administered activated charcoal is the recommended treatment for many poisonings. However, ingestion of central nervous system depressants resulting in loss of protective airway reflexes may result in pulmonary aspiration of activated charcoal. Although activated charcoal has been reported to be an inert substance, evidence suggests that pulmonary aspiration of charcoal is associated with lung edema formation and pulmonary compromise. This study tested the hypothesis that intratracheal instillation of activated charcoal disrupts the integrity of the lung microvascular barrier. METHODS: The capillary filtration coefficient (Kf,c), a sensitive measure of lung microvascular permeability, was determined isogravimetrically prior to and after intratracheal instillation of activated charcoal 0.04 g/kg (12% weight/vol solution, pH 7.4) or an equal volume of sterile water in isolated, perfused rat lungs. Arterial blood gas analysis was determined prior to and after tracheal instillation of activated charcoal or sterile water in a separate group of animals. RESULTS: Intratracheal instillation of activated charcoal resulted in a significant increase in pulmonary microvascular permeability compared to lungs treated with sterile water or control lungs (delta Kf,c = +0.21 +/- 0.076; -0.014 +/- 0.04; and -0.041 +/- 0.02 mL/min/cm H2O/100 g lung tissue, respectively, p < 0.05 ANOVA). There was no significant difference in baseline blood gases in the 3 experimental groups. There was a significant decrease in arterial Po2, bicarbonate, and pH in animals administered activated charcoal compared to time-matched controls and animals administered sterile water. CONCLUSIONS: Intratracheal instillation of activated charcoal is associated with a significant increase in lung microvascular permeability and arterial blood gas derangements. The effects of activated charcoal on pulmonary microvascular barrier integrity may contribute to the lung edema formation and pulmonary compromise observed following clinical aspiration of activated charcoal.

Neutrophil elastase promotes lung microvascular injury and proteolysis of endothelial cadherins.

Intestinal ischemia-reperfusion (I-R) is associated with lung injury and the acute respiratory distress syndrome. The hypothesis of this study was that intestinal I-R activates circulating neutrophils to promote elastase-mediated lung injury. Isolated rat lungs were perfused with blood or plasma obtained after intestinal I-R, and lung neutrophil retention and injury and bronchoalveolar lavage (BAL) elastase were measured. Perfusion with I-R blood caused lung neutrophil accumulation and injury and increased BAL elastase. These effects were attenuated by the elastase inhibitor L-658758. Interference with neutrophil adherence before gut reperfusion blocked BAL elastase accumulation. The role of endothelial junction proteins (cadherins) in I-R-elicited lung damage was also evaluated. Activated human neutrophils proteolyzed cadherins in human umbilical vein endothelial cells. Furthermore, plasma of patients with acute respiratory distress syndrome contained soluble cadherin fragments. The results of this study suggest that the elastase released by systemically activated neutrophils contributes to lung neutrophil accumulation and pulmonary microvascular injury. Elastase-mediated proteolysis of endothelial cell cadherins may represent the mechanism through which lung microvascular integrity is disrupted after intestinal I-R.

Isolation of chromatic and achromatic mechanisms: a new approach.

The purpose of this study was to optimise the testing paradigm for isolating the contributions of chromatic and achromatic mechanisms to the human spectral sensitivity function. Spectral sensitivity was determined for a test spot size of 1.2 deg presented with various spatial and temporal masks on a large, 10 deg background field of moderate intensity (1000 td) and colour temperature, CT = 2700 K. Sinusoidal temporal presentation (1 Hz) and a masking annulus of between 3 and 10 min of arc surrounding the test spot, was found to be most effective in separating chromatic from achromatic mechanisms. Square-wave (1 Hz) temporal presentation combined with the annulus was slightly less selective. The presence of the annulus did not affect the shape of flicker detection at 25 Hz which is a measure of the luminosity (achromatic) spectral sensitivity function.

Funding strategies for emergency medicine research.

The importance of adequate funding for sustaining research efforts cannot be overemphasized. This article addresses funding strategies for emergency physicians, including the necessity of establishing a research track record, developing a well-written grant proposal, and anticipating the grant review process. Funding sources are reviewed with an emphasis on federal institute support and private foundations (including the Emergency Medicine Foundation) in the United States. Sources of current grant support information available from the Internet are provided. Recommendations for enhancing research funding in emergency medicine (EM) are made, including enhancement of formal research training, promotion of EM research and investigators, federal study section membership, and collaboration with established investigators.