Multiscale, multidomain analysis of microvascular flow dynamics.

NEW FINDINGS: What is the topic of this review? We describe a range of techniques in the time, frequency and information domains and their application alone and together for the analysis of blood flux signals acquired using laser Doppler fluximetry. What advances does it highlight? This review highlights the idea of using quantitative measures in different domains and scales to gain a better mechanistic understanding of the complex behaviours in the microcirculation. ABSTRACT: To date, time- and frequency-domain metrics of signals acquired through laser Doppler fluximetry have been unable to provide consistent and robust measures of the changes that occur in the microcirculation in healthy individuals at rest or in response to a provocation, or in patient cohorts. Recent studies have shown that in many disease states, such as metabolic and cardiovascular disease, there appears to be a reduction in the adaptive capabilities of the microvascular network and a consequent reduction in physiological information content. Here, we introduce non-linear measures for assessing the information content of fluximetry signals and demonstrate how they can yield deeper understanding of network behaviour. In addition, we show how these methods may be adapted to accommodate the multiple time scales modulating blood flow and how they can be used in combination with time- and frequency-domain metrics to discriminate more effectively between the different mechanistic influences on network properties.

Using high resolution X-ray computed tomography to create an image based model of a lymph node.

Lymph nodes are an important part of the immune system. They filter the lymphatic fluid as it is transported from the tissues before being returned to the blood stream. The fluid flow through the nodes influences the behaviour of the immune cells that gather within the nodes and the structure of the node itself. Measuring the fluid flow in lymph nodes experimentally is challenging due to their small size and fragility. In this paper, we present high resolution X-ray computed tomography images of a murine lymph node. The impact of the resulting visualized structures on fluid transport are investigated using an image based model. The high contrast between different structures within the lymph node provided by phase contrast X-ray computed tomography reconstruction results in images that, when related to the permeability of the lymph node tissue, suggest an increased fluid velocity through the interstitial channels in the lymph node tissue. Fluid taking a direct path from the afferent to the efferent lymphatic vessel, through the centre of the node, moved faster than the fluid that flowed around the periphery of the lymph node. This is a possible mechanism for particles being moved into the cortex.

Phase contrast synchrotron radiation computed tomography of muscle spindles in the mouse soleus muscle.

Muscle spindles are skeletal muscle sensory organs involved in the sensation of position and movement of the body. We have explored the capability of phase contrast computed tomography to visualise muscle spindles in murine skeletal muscle. In particular, we have validated the visualisation of nerve fibres through phase contrast computed tomography using light microscopy on stained histological sections. We further present the first three-dimensional visualisation of muscle spindles in mouse soleus skeletal muscle in conjunction with the neurovascular bundle associated with it.

Developmental aspects of a life course approach to healthy ageing.

We examine the mechanistic basis and wider implications of adopting a developmental perspective on human ageing. Previous models of ageing have concentrated on its genetic basis, or the detrimental effects of accumulated damage, but also have raised issues about whether ageing can be viewed as adaptive itself, or is a consequence of other adaptive processes, for example if maintenance and repair processes in the period up to reproduction are traded off against later decline in function. A life course model places ageing in the context of the attainment of peak capacity for a body system, starting in early development when plasticity permits changes in structure and function induced by a range of environmental stimuli, followed by a period of decline, the rate of which depends on the peak attained as well as the later life conditions. Such path dependency in the rate of ageing may offer new insights into its modification. Focusing on musculoskeletal and cardiovascular function, we discuss this model and the possible underlying mechanisms, including endothelial function, oxidative stress, stem cells and nutritional factors such as vitamin D status. Epigenetic changes induced during developmental plasticity, and immune function may provide a common mechanistic process underlying a life course model of ageing. The life course trajectory differs in high and low resource settings. New insights into the developmental components of the life course model of ageing may lead to the design of biomarkers of later chronic disease risk and to new interventions to promote healthy ageing, with important implications for public health.

Skeletal muscle morphology in sarcopenia defined using the EWGSOP criteria: findings from the Hertfordshire Sarcopenia Study (HSS).

BACKGROUND: Sarcopenia is defined as the loss of muscle mass and function with age and is associated with decline in mobility, frailty, falls and mortality. There is considerable interest in understanding the underlying mechanisms. Our aim was to characterise muscle morphology changes associated with sarcopenia among community dwelling older men. METHODS: One hundred and five men aged 68-76 years were recruited to the Hertfordshire Sarcopenia Study (HSS) for detailed characterisation of muscle including measures of muscle mass, strength and function. Muscle tissue was obtained from a biopsy of the vastus lateralis for 99 men and was processed for immunohistochemical studies to determine myofibre distribution and area, capillarisation and satellite cell (SC) density. RESULTS: Six (6 %) men had sarcopenia as defined by the European Working Group on Sarcopenia in Older People (EWGSOP) criteria. These men had lower SC density (1.7 cells/mm(2) vs 3.8 cells/mm(2), p = 0.06) and lower SC/fibre ratio (0.02 vs 0.06, p = 0.06) than men without sarcopenia. Although men with sarcopenia tended to have smaller myofibres and lower capillary to fibre ratio, these relationships were not statistically significant. CONCLUSION: We have shown that there may be altered muscle morphology parameters in older men with sarcopenia. These results have the potential to help identify cell and molecular targets for therapeutic intervention. This work now requires extension to larger studies which also include women.

Prostaglandin E2 and nitric oxide mediate the acute inflammatory (erythemal) response to topical 5-aminolaevulinic acid photodynamic therapy in human skin.

BACKGROUND: Topical 5-aminolaevulinic acid photodynamic therapy (5-ALA-PDT) causes a clinical inflammatory response in human skin. While histamine mediates the immediate reaction, the mediators of the prolonged erythema are unknown. OBJECTIVES: To look for involvement of the proinflammatory mediators prostaglandin (PG)E2 and nitric oxide (NO) in topical PDT-induced erythema in human skin. METHODS: A series of studies was performed in healthy volunteers (n = 35). Following definition of the erythemal time course and dose response to 5-ALA-PDT, duplicate 5-ALA dose series were iontophoresed into the skin of each ventral forearm and exposed to 100 J cm(-2) broadband red light. Within subject, arms were randomized to control, or treatment with the cyclooxygenase and NO synthase inhibitors indometacin and Nω -nitro-l-arginine methyl ester (l-NAME), respectively, and the impact on 5-ALA-PDT-induced erythema was quantified. Additionally, release of PGE2 and NO was directly assessed by sampling dermal microdialysate at intervals following 5-ALA-PDT administration. RESULTS: A 5-ALA dose-related delayed erythema occurred by 3 h (r = 0·97, P < 0·01), with erythema persisting to 48 h post-PDT. Topical indometacin applied immediately post-PDT reduced the slope of erythemal response at 3 h and 24 h (P < 0·05). Intradermal injection of l-NAME into 5-ALA-PDT-treated sites reduced the slope of response at 24 h post-PDT (P < 0·001), while significantly inhibiting erythema from 3 h to 48 h post-PDT (P < 0·01). Analysis of dermal microdialysate showed release of NO and PGE2 following treatment. CONCLUSIONS: Topical 5-ALA-PDT upregulates PGE2 and NO in human skin, where they play a significant role in the clinical inflammatory response. The potential relevance of these mediators to PDT in human cutaneous pathology warrants study.

64×64 pixel smart sensor array for laser Doppler blood flow imaging.

What is believed to be the first fully integrated two-dimensional complementary metal oxide semiconductor (CMOS) imaging array for laser Doppler blood flow imaging is demonstrated. The sensor has 64×64 pixels and includes both analog and digital on-chip processing electronics. This offers several potential advantages over commercial sensors as the processing is tailored to the signals of interest and the data bottleneck that exists between the sensor and processing electronics is overcome. To obtain a space efficient design over 64×64 pixels means that standard processing electronics used off-chip cannot be implemented. Images of both simulated blood flow responses and a blood flow occlusion test demonstrate the capability.

Metabolic regulation during constant moderate physical exertion in extreme conditions in Type 1 diabetes.

BACKGROUND: Constant moderate intensity physical exertion in humid environments at altitude poses a considerable challenge to maintaining euglycaemia with Type 1 diabetes. Blood glucose concentrations and energy expenditure were continuously recorded in a person trekking at altitude in a tropical climate to quantify changes in glucose concentrations in relation to energy expenditure. CASE REPORT: Blood glucose concentrations and energy expenditure were continuously monitored with a Guardian® real-time continuous glucose monitoring system (CGMS) and a SenseWear® Pro3 armband (BodyMedia Inc., USA), in a 27-year-old woman with Type 1 diabetes, during her climb up Mount Kinabalu in Borneo (c. 4095 m). Comparative control data from the same person was collected in the UK (temperate climate at sea level) and Singapore (tropical climate at sea level). Maximum physical effort during the climb was < 60% VO(2MAX) (maximal oxygen consumption). Mean daily calorific intakes were 2300 kcal (UK), 2370 kcal (Singapore) and 2274 kcal (Mount Kinabalu), and mean daily insulin doses were 54 U (UK), 40 U (Singapore) and 47 U (Mount Kinabalu). Despite markedly increased energy expenditure during the climb [4202 kcal (Mount Kinabalu) vs. 2948 kcal (UK) and 2662 kcal (Singapore)], mean blood glucose was considerably higher during the trek up Mount Kinabalu [13.2 ± 5.9 mmol/l, vs. 7.9 ± 3.8 mmol/l (UK) and 8.6 ± 4.0 mmol/l (Singapore)]. CONCLUSION: Marked unexpected hyperglycaemia occurred while trekking on Mount Kinabalu, despite similar calorie consumption and insulin doses to control conditions. Because of the risk of unexpected hyperglycaemia in these conditions, we recommend that patients embarking on similar activity holidays undertake frequent blood glucose monitoring.

Skeletal muscle microvascular exchange capacity is associated with hyperglycaemia in subjects with central obesity.

AIMS: Poor glycaemic control is associated with increased risk of microvascular disease in various organs including the eye and kidney, but the relationship between glycated haemoglobin (HbA(1c)) and microvascular function in skeletal muscle has not been described. We tested the association between HbA(1c) and a measure of microvascular exchange capacity (K(f)) in skeletal muscle in people with central obesity at risk of developing Type 2 diabetes. METHODS: Microvascular function was measured in 28 women and 19 men [mean (+/- sd) age 51 +/- 9 years] with central obesity who did not have diabetes. We estimated insulin sensitivity by hyperinsulinaemic-euglycaemic clamp, visceral and total fatness by magnetic resonance imaging, fitness (VO(2) max by treadmill testing), physical activity energy expenditure [metabolic equivalents of tasks (METS) by use of the SenseWear Pro armband] and skeletal muscle microvascular exchange capacity (K(f)) by venous occlusion plethysmography. RESULTS: In regression modelling, age, sex and fasting plasma glucose accounted for 30.5% of the variance in HbA(1c) (r(2) = 0.31, P = 0.001). Adding K(f) to this model explained an additional 26.5% of the variance in HbA(1c) (r(2) = 0.57, P = 0.0001 and K(f) was strongly and independently associated with HbA(1c) (standardized B coefficient -0.45 (95% confidence interval -0.19, -0.06), P = 0.001). CONCLUSIONS: We found a strong negative independent association between a measure of skeletal muscle microvascular exchange capacity (K(f)) and HbA(1c). K(f) was associated with almost as much of the variance in HbA(1c) as fasting plasma glucose.

Image-based modelling of skeletal muscle oxygenation.

The supply of oxygen in sufficient quantity is vital for the correct functioning of all organs in the human body, in particular for skeletal muscle during exercise. Disease is often associated with both an inhibition of the microvascular supply capability and is thought to relate to changes in the structure of blood vessel networks. Different methods exist to investigate the influence of the microvascular structure on tissue oxygenation, varying over a range of application areas, i.e. biological in vivo and in vitro experiments, imaging and mathematical modelling. Ideally, all of these methods should be combined within the same framework in order to fully understand the processes involved. This review discusses the mathematical models of skeletal muscle oxygenation currently available that are based upon images taken of the muscle microvasculature in vivo and ex vivo Imaging systems suitable for capturing the blood vessel networks are discussed and respective contrasting methods presented. The review further informs the association between anatomical characteristics in health and disease. With this review we give the reader a tool to understand and establish the workflow of developing an image-based model of skeletal muscle oxygenation. Finally, we give an outlook for improvements needed for measurements and imaging techniques to adequately investigate the microvascular capability for oxygen exchange.

Investigation of microvascular morphological measures for skeletal muscle tissue oxygenation by image-based modelling in three dimensions.

The supply of oxygen in sufficient quantity is vital for the correct functioning of all organs in the human body, especially for skeletal muscle during exercise. Traditionally, microvascular oxygen supply capability is assessed by the analysis of morphological measures on transverse cross-sections of muscle, e.g. capillary density or capillary-to-fibre ratio. In this work, we investigate the relationship between microvascular structure and muscle tissue oxygenation in mice. Phase contrast imaging was performed using synchrotron radiation computed tomography (SR CT) to visualize red blood cells (RBCs) within the microvasculature in mouse soleus muscle. Image-based mathematical modelling of the oxygen diffusion from the RBCs into the muscle tissue was subsequently performed, as well as a morphometric analysis of the microvasculature. The mean tissue oxygenation was then compared with the morphological measures of the microvasculature. RBC volume fraction and spacing (mean distance of any point in tissue to the closest RBC) emerged as the best predictors for muscle tissue oxygenation, followed by length density (summed RBC length over muscle volume). The two-dimensional measures of capillary density and capillary-to-fibre ratio ranked last. We, therefore, conclude that, in order to assess the states of health of muscle tissue, it is advisable to rely on three-dimensional morphological measures rather than on the traditional two-dimensional measures.

Ultraviolet-B-induced erythema is mediated by nitric oxide and prostaglandin E2 in combination.

Ultraviolet-B-induced erythema (one, two, or four times the minimal erythema dose) was reduced but not abolished by application of 1% indomethacin gel immediately after irradiation of human skin. Continuous synthesis of prostaglandins is reflected by similar levels of indomethacin-mediated inhibition of erythema at any time within 48 h after irradiation. Repeated applications of indomethacin did not increase the inhibition. Twenty-four hours after irradiation with four minimal erythema doses, mean prostaglandin E2 levels in suction blisters were 27.2 ng per ml (SEM 11) compared with 8.6 ng per ml in unirradiated skin (n = 25; p < 0.01). Prosta glandin E2 levels in dermal tissues, sampled by microdialysis (depth 0.6 +/- 0.1 mm), were 310 pg per ml (SEM 123) and 237 pg per ml (SEM 88) in irradiated and unirradiated skin, respectively (n = 7, n.s.). Nitric oxide also made a significant contribution to ultraviolet-B-induced erythema. Ultraviolet erythema was inhibited by L-NAME in a dose-related fashion with 2 mM L-NAME causing total abolition of the response. L-NAME was effective at all time points up to 48 h suggesting that NO was produced continuously. NO was undetectable in suction blister fluid but in dermal microdialysate NO was present at 44.3 ng per ml (SEM 6.2) following ultraviolet B compared with 26.0 ng per ml (SEM 8.0) in unirradiated skin (p < 0.05), approximately 1000 times the molar concentration of prostaglandin E2. These findings confirm prostaglandin E2 and NO to be mediators of ultraviolet-induced erythema. They also show that there is prolonged synthesis of both mediators within the erythemal response and that synthesis of NO is induced by lower doses of ultraviolet B compared with that of prostaglandin E2.

Inhibition by glucocorticoids of the mast cell-dependent weal and flare response in human skin in vivo.

1. This study examines the relative contributions made by inhibition of mast cell degranulation, reduction of mast cell recruitment and maturation, and lowering the responsiveness of the vasculature to histamine, in the inhibition by glucocorticoids of the weal and flare in human skin. 2. One forearm of healthy human volunteers was treated for 24 h (n=6) or daily for 21 days (n=10) with 0.05% clobetasol propionate. The other arm served as control. Weal and flare responses were elicited by intradermal injection of 20 microl of 0.3 mM codeine. The areas of the responses were measured using scanning laser Doppler imaging. Microdialysis was used to assess histamine release. Mast cell numbers and tissue histamine content were assessed in 4-mm punch biopsies. Histamine (20 microl of 1 microM i.d.) was used to assess the status of the vasculature. 3. No significant effects were seen at 24 h. At 21 days, clobetasol reduced the areas of the codeine-induced weal and flare responses by 59 and 58% respectively (both P=0.006). Mast cell numbers were reduced by 47%, (P=0.014) and total tissue histamine content by 52% (P=0.006). Codeine-induced histamine release was reduced by 44% (P=0.022). The weal, but not the flare, induced by histamine was significantly inhibited (P=0.019). Echography revealed a 15% thinning of the skin by clobetasol. 4. These results demonstrate that reduction of the weal and flare responses to codeine following clobetasol treatment, results primarily from reduced mast cell numbers and tissue histamine content rather than inhibition by corticosteroids of mast cell degranulation.

Changing sensitivity to H1 and H2 receptor antagonists in the growing vasculature.

1. Resting albumin efflux is greater from immature vessels than from mature and this is associated with a greater blood flow in immature vessels. 2. Both resting blood flow and albumin efflux in immature vessels are reduced by an H2 antagonist. 3. Histamine produces a greater albumin efflux from mature vessels than from immature and the associated increase in blood flow in response to histamine was also greater in mature vessels. 4. Blood flow and permeability increase produced by histamine are separately mediated via H2 and H1 receptors respectively in mature vessels. 5. In immature vessels, H2 receptors appear to mediate both responses.

The microvasculature: a target for nutritional programming and later risk of cardio-metabolic disease.

There is compelling evidence that microvascular deficits affecting multiple tissues and organs play an important role in the aetiopathogenesis of cardio-metabolic disease. Furthermore, both in humans and animal models, deficits in small vessel structure and function can be detected early, often before the onset of macrovascular disease and the development of end-organ damage that is common to hypertension and obesity-associated clinical disorders. This article considers the growing evidence for the negative impact of an adverse maternal diet on the long-term health of her child, and how this can result in a disadvantageous vascular phenotype that extends to the microvascular bed. We describe how structural and functional modifications in the offspring microcirculation during development may represent an important and additional risk determinant to increase susceptibility to the development of cardio-metabolic disease in adult life and consider the cell-signalling pathways associated with endothelial dysfunction that may be 'primed' by the maternal environment. Published studies were identified that reported outcomes related to the microcirculation, endothelium, maternal diet and vascular programming using NCBI PubMed.gov, MEDLINE and ISI Web of Science databases from 1980 until April 2013 using pre-specified search terms. Information extracted from over 230 original reports and review articles was critically evaluated by the authors for inclusion in this review.

Design and rationale of the WELCOME trial: A randomised, placebo controlled study to test the efficacy of purified long chainomega-3 fatty acid treatment in non-alcoholic fatty liver disease [corrected].

BACKGROUND: Non-alcoholic fatty liver disease (NAFLD) represents a range of liver conditions from simple fatty liver to progressive end stage liver disease requiring liver transplantation. NAFLD is common in the population and in certain sub groups (e.g. type 2 diabetes) up to 70% of patients may be affected. NAFLD is not only a cause of end stage liver disease and hepatocellular carcinoma, but is also an independent risk factor for type 2 diabetes and cardiovascular disease. Consequently, effective treatments for NAFLD are urgently needed. OBJECTIVES: The WELCOME study is testing the hypothesis that treatment with high dose purified long chain omega-3 fatty acids will have a beneficial effect on a) liver fat percentage and b) two histologically validated algorithmically-derived biomarker scores for liver fibrosis. DESIGN: In a randomised double blind placebo controlled trial, 103 participants with NAFLD were randomised to 15-18months treatment with either 4g/day purified long chain omega-3 fatty acids (Omacor) or 4g/day olive oil as placebo. Erythrocyte percentage DHA and EPA enrichment (a validated proxy for hepatic enrichment) was determined by gas chromatography. Liver fat percentage was measured in three discrete liver zones by magnetic resonance spectroscopy (MRS). We also measured body fat distribution, physical activity and a range of cardiometabolic risk factors. METHODS: Recruitment started in January 2010 and ended in June 2011. We identified 178 potential participants, and randomised 103 participants who met the inclusion criteria. The WELCOME study was approved by the local ethics committee (REC: 08/H0502/165; www.clinicalTrials.gov registration number NCT00760513).

Diastolic function is strongly and independently associated with cardiorespiratory fitness in central obesity.

Cardiorespiratory fitness [maximal O2 consumption (VO2max)] is an independent risk factor for type 2 diabetes; but in individuals at risk, factors influencing VO2max are poorly understood. We tested the hypothesis that VO2max is associated with diastolic function [subendocardial variability ratio (SEVR), %], as diastolic function influences myocardial perfusion. We studied 47 men and women with central obesity without diabetes. We measured fitness (VO2max) by treadmill testing and diastolic function (SEVR%) by pulse-wave analysis. We measured other factors influencing this relationship: insulin sensitivity [whole body glucose uptake-to-insulin concentration ratio (M/I)] by hyperinsulinemic euglycemic clamp, fatness by MR imaging and dual-energy X-ray absorptiometry, physical activity energy expenditure (metabolic equivalents of tasks) by the Sensewear Pro2 device, and muscle microvascular exchange capacity (capillary filtration coefficient) by venous plethysmography. Mean age of the subjects was 51+/-9 (SD) yr. VO2max was associated with SEVR% (r=0.50, P=0.001), fatness (r=-0.39, P=0.008), and HbA1c (r=-0.35, P=0.018), but not with whole body glucose uptake-to-insulin concentration ratio, metabolic equivalents of tasks, or capillary filtration coefficient. In regression modeling with age, sex, fatness, and SEVR% as explanatory variables, only age, sex, and SEVR% were independently associated with VO2max (SEVR%--standardized B coefficient=0.37, 95% confidence interval=0.003-0.18, P=0.007). This model identified 46% of the variance in VO2max (R2=0.46, P=0.0001). There was a strong, independent association between VO2max and a measure of diastolic function in sedentary individuals with central obesity.

Role of nitric oxide in the regulation of microvascular perfusion in human skin in vivo.

1. Nitric oxide (NO) concentrations were measured in dialysate from healthy human skin, in vivo, both at rest and during the inflammatory response to intradermal histamine or bradykinin. Changes in dialysate NO concentration, measured by electrochemical detection, were related to changes in dermal vascular perfusion, measured using scanning laser Doppler imaging. 2. Basal NO concentration in dermal microdialysate was 0.60 +/- 0.14 microM (mean +/- s.e.m.). Following the intradermal injection of histamine, a transient, time-dependent increase in NO concentration was measured in areas of skin incorporating the weal and in others incorporating the flare. The increase in NO concentration was associated with an increase in dialysate cGMP concentration in both the weal and flare areas. 3. Addition of N G-nitro-l-arginine-methyl ester (L-NAME, 5 mM) to the probe perfusate resulted in an inhibition of the histamine-induced increase in NO and cGMP. Moreover, the reduction in dialysate NO concentration was associated with a reduction in dermal vascular flux, both under basal conditions and within the weal and flare response. 4. These results demonstrate, by the use of microdialysis, that vasoactive mediators can be measured in healthy human skin in vivo. They provide direct evidence that endogenous concentration of NO increases during the inflammatory weal and flare response to histamine and that the increase in dermal NO concentration is associated with increases in cGMP concentration and dermal vascular perfusion, thus confirming a role for NO in vasoregulation in human skin.

Human skin mast cells: in vitro and in vivo studies.

OBJECTIVE: This short review surveys our current knowledge on the development and heterogeneity of human mast cells, the distribution of mast cells within human skin and the properties of human skin mast cells both in vitro and in vivo. It also examines the effects of antihistamines in the wheal-and-flare response in the skin provoked by bradykinin. RESULTS: Mast cells derive from mononuclear precursor cells which undergo their final phase of their differentiation in the tissues. In normal skin, mast cells, which are primarily of the MC(TC) subtype, occur in the greatest density in the superficial dermal zone. Like all other mast cells, human skin mast cells bind IgE with high affinity to specific FcepsilonRI receptors, but unlike those from lung, tonsils, adenoids or intestine, they also express the C5a receptor (CD88) and activation sites for substance P, VIP, somatostatin, and compound 48/80. Both IgE-dependent stimulation by activating tyrosine kinases, and non-immunologic stimulation by activating G-proteins induce a characteristic compound exocytosis resulting in the liberation of the preformed mediators. Production of prostaglandin D2 and leukotriene C4, however, occurs only with IgE-dependent stimulation. In vivo, dermal microdialysis and scanning laser Doppler imaging have been used to assess the role of histamine in the wheal-and-flare response. These techniques were also used to show that low concentrations of intradermal bradykinin release negligible quantities of histamine. The results showed that although the resultant flare was inhibitable by antihistamines, low concentrations of bradykinin released negligible quantities of histamine. This suggests a potentially novel mechanism of action of antihistamines that requires further detailed investigation.

Investigation into the mechanisms by which nedocromil sodium, frusemide and bumetanide inhibit the histamine-induced itch and flare response in human skin in vivo.

BACKGROUND: In a previous study, iontophoresis of nedocromil sodium into human skin in vivo was shown to reduce histamine-induced itch and flare. In asthma, the Na+/K+/2Cl- cotransporter inhibitors, frusemide and bumetanide, have been reported to have many similar actions to nedocromil sodium. OBJECTIVE: To compare the effects of these drugs in the histamine-induced itch, flare and weal response in human skin in vivo and elucidate their site of action. METHODS: Nedocromil sodium, frusemide bumetanide and reversed osmosis water (control), were introduced by iontophoresis into the forearm skin of 10 volunteers in each of two single-blind studies. In study 1, histamine (20 microL of 100 microM) or vehicle was injected into the area of iontophoresis 10 min later. In study 2, histamine or vehicle was injected 5 mm outside the area of iontophoresis so the flare developed over the area of iontophoresis. Itch was scored on a visual analogue scale every 20 s for 5 min, flare areas were assessed using scanning laser Doppler imaging up to 10 min and weal was assessed by planimetry at 10 min. RESULTS: In study 1, nedocromil sodium, frusemide and bumetanide reduced itch scores by 36%, 48% and 34%, respectively, and flare areas by 17%, 26% and 15% respectively (all P<0.05). Weal areas and blood flux in the flare were unaffected. In study 2, itch scores, flare areas and weal areas were not inhibited. Also, blood flux values in areas of drug and water iontophoresis were not different. CONCLUSION: This study has provided evidence to support the hypothesis that nedocromil sodium, frusemide and bumetanide inhibit sensory nerve activation to reduce the itch and flare responses induced by histamine in human skin in vivo. It is likely that inhibition of a Na+/K+/2Cl- cotransporter in the sensory nerve membrane is a possible mechanism of action.