Dermal Blood Flow Research Articles

P116 - Increasing dermal blood flow with photochemical NO release from topical formulations

P116 - Increasing dermal blood flow with photochemical NO release from topical formulations

EHMTI-0315. AMG 334, the first potent and selective human monoclonal antibody antagonist against the CGRP receptor

Results AMG 334 is a potent inhibitor of [125I]-CGRP binding to the human CGRP receptor with a Ki of 0.02 nM. It exhibited full inhibition of CGRP-stimulated cAMP production with an IC50 of 2.3 nM in cell-based functional assays. Potency of AMG 334 at the cyno CGRP receptor is similar to that at the human receptor, but with significant reduced potency at dog, rabbit and rat receptors. AMG 334 also demonstrates > 5000-fold selectivity over other closely related receptors in the family. The receptor kinetics studies using [125I]-AMG 334 reveals a dissociation t1/2 off of 67 min. In the cyno study, AMG 334 produces a significant and sustained inhibitory effect on capsaicin-induced increase in dermal blood flow.

PM175 Reproducibility of dermal microvascular blood flow changes in response to reactive and thermal hyperaemia utilising laser skin doppler

O ST E R A B ST R A C T S adiposity, smoking, systolic blood pressure, and metabolic syndrome were inversely associated with endothelial function after adjustment for age and sex (Table). However, there were no associations between early life growth and endothelial function. Conclusion: These are the first prospective data from India showing that cardiometabolic risk factors, but not early life growth, are inversely associated with endothelial function. The high cardiometabolic risk factor burden in this cohort may have attenuated any potential associations between early life growth and endothelial function. Disclosure of Interest: None Declared

Review: Tissue Optical Clearing Window for Blood Flow Monitoring

The tissue optical clearing (TOC) technique could significantly improve the biomedical optical imaging depth, but most current investigations are limited to in vitro studies. For in vivo applications, the TOC method must provide a rapid treatment process, sufficient transparency, and safety for animals, which makes it more difficult. Recently developed innovative optical clearing methods for in vivo use show great potential for enhancing the contrast and resolution of laser speckle contrast imaging (LSCI) for blood flow monitoring. This paper gives an overview of recent progress in the use of TOC for vascular visualization with LSCI. First, the principle of TOC-induced improvement of LSCI and a quantitative analysis method for evaluating the improvement are described briefly. Second, the paper introduces transparent windows, including various skin windows and a cranial window, that permit LSCI to monitor dermal or cortical blood flow, respectively, with high resolution and contrast. Third, preliminary investigations of the safety of TOC demonstrate that the transparent skin window is switchable, which enables LSCI to repeatedly image blood flow. However, research on in vivo TOC is currently less advanced than that on in vitro TOC. Future work should focus on developing a highly effective, safe method and extending its applications.

Sugar-Induced Skin Optical Clearing: From Molecular Dynamics Simulation to Experimental Demonstration

The tissue optical clearing technique can significantly improve the quality of biomedical optical imaging. Previous investigations demonstrated that alcohols are excellent optical clearing agents for in vitro skin because they have hydroxyl. However, sugars with hydroxyl groups have received little attention. Here, three sugars, fructose, glucose, and ribose, were investigated using a molecular dynamics simulation and in vitro experiments. The simulated results revealed that fructose and ribose have the highest and lowest optical clearing potential, respectively, according to their propensity to form hydrogen bonds and bridges. The resolutions through skin samples after 24 min of immersion in fructose, glucose, or ribose solutions were quantitatively calculated using a USAF target and reached 14.3 ± 1.2 μm, 27.3 ± 2.4 μm, and 39.6 ± 4.1 μm, respectively, which are in accordance with the theoretical prediction. Unfortunately, topical fructose treatment of in vivo rat skin could not induce excellent optical clearing. Thus, a chemical penetration enhancer was used for in vivo studies. A comparison of laser speckle imaging of dermal blood flow after topical treatment with fructose, a penetration enhancer, and a mixture of the two to rat skin in vivo demonstrated that the mixture causes the largest increase in the resolution and contrast.

Development of a novel antibody to calcitonin gene-related peptide for the treatment of osteoarthritis-related pain

Investigate a role for calcitonin gene-related peptide (CGRP) in osteoarthritis (OA)-related pain. Neutralizing antibodies to CGRP were generated de novo. One of these antibodies, LY2951742, was characterized invitro and tested in pre-clinical invivo models of OA pain. LY2951742 exhibited high affinity to both human and rat CGRP (KD of 31 and 246pM, respectively). The antibody neutralized CGRP-mediated induction of cAMP in SK-N-MC cells invitro and capsaicin-induced dermal blood flow in the rat. Neutralization of CGRP significantly reduced pain behavior as measured by weight bearing differential in the rat monoiodoacetate model of OA pain in a dose-dependent manner. Moreover, pain reduction with neutralization of CGRP occurred independently of prostaglandins, since LY2951742 and NSAIDs worked additively in the NSAID-responsive version of the model and CGRP neutralization remained effective in the NSAID non-responsive version of the model. Neutralization of CGRP also provided dose-dependent and prolonged (>60 days) pain reduction in the rat meniscal tear model of OA after only a single injection of LY2951742. LY2951742 is a high affinity, neutralizing antibody to CGRP. Neutralization of CGRP is efficacious in several OA pain models and works independently of NSAID mechanisms of action. LY2951742 holds promise for the treatment of pain in OA patients.

Development of an experimental model to study trigeminal nerve-mediated vasodilation on the human forehead

During migraine, trigeminal sensory nerve terminals release calcitonin gene-related peptide (CGRP), inducing nociception and vasodilation. Applied on the skin, capsaicin activates the transient receptor potential vanilloid type 1 (TRPV1) channel and releases CGRP from sensory nerve terminals, thus increasing dermal blood flow (DBF). Using capsaicin application and electrical stimulation of the forehead skin, a trigeminal nerve-innervated dermatome, we aimed to develop a model to measure trigeminal nerve-mediated vasodilation in humans. Using laser Doppler imaging, forehead DBF responses to application of capsaicin (0.06 mg/ml and 6.0 mg/ml) and saline, with and without iontophoresis, were studied in healthy subjects. The within-subject coefficient of variation (WCV) of repeated DBF measurements was calculated to assess reproducibility. Maximal DBF responses to 6.0 mg/ml capsaicin with and without iontophoresis did not differ (Emax 459 ± 32 and 424 ± 32 arbitrary units (a.u.), WCV 6 ± 4%). In contrast, DBF responses to 0.06 mg/ml capsaicin were significantly larger with than without iontophoresis (Emax 307 ± 60 versus 187 ± 21 a.u., WCV 21 ± 13%). Saline with iontophoresis significantly increased DBF (Emax: 245 ± 26 a.u, WCV 11 ± 8%), while saline application without iontophoresis did not affect DBF. Topical application of capsaicin and electrical stimulation induce reproducible forehead DBF increases and therefore are suitable to study trigeminal nerve-mediated vasodilation in humans.

Nitric oxide-releasing poly(vinyl alcohol) film for increasing dermal vasodilation

Pathological conditions associated with the impairment of nitric oxide (NO) production in the vasculature, such as Raynaud's syndrome and diabetic angiopathy, have stimulated the development of new biomaterials capable of delivering NO topically. With this purpose, we modified poly(vinyl-alcohol) (PVA) by chemically crosslinking it via esterification with mercaptosuccinic acid. This reaction allowed the casting of sulfhydrylated PVA (PVA-SH) films. Differential scanning calorimetry and X-ray diffractometry showed that the crosslinking reaction completely suppressed the crystallization of PVA, leading to a non-porous film with a homogeneous distribution of -SH groups. The remaining free hydroxyl groups in the PVA-SH network conferred partial hydrophylicity to the material, which was responsible for a swelling degree of ca. 110%. The PVA-SH films were subjected to an S-nitrosation reaction of the –SH groups, yielding a PVA containing S-nitrosothiol groups (PVA-SNO). Amperometric and chemiluminescence measurements showed that the PVA-SNO films were capable of releasing NO spontaneously after immersion in physiological medium. Laser Doppler-flowmetry, used to assess the blood flow in the dermal microcirculation, showed that the topical application of hydrated PVA-SNO films on the health skin led to a dose- and time-dependent increase of more than 5-fold in the dermal baseline blood flow in less than 10min, with a prolonged action of more than 4h during continuous application. These results show that PVA-SNO films might emerge as a new material with potential for the topical treatment of microvascular skin disorders.

Combination of HIF-1α gene transfection and HIF-1-activated bone marrow-derived angiogenic cell infusion improves burn wound healing in aged mice

Impaired burn wound healing in the elderly represents a major clinical problem. Hypoxia-inducible factor-1 (HIF-1) is a transcriptional activator that orchestrates the cellular response to hypoxia. Its actions in dermal wounds promote angiogenesis and improve healing. In a murine burn wound model, aged mice had impaired wound healing associated with reduced levels of HIF-1. When gene therapy with HIF-1 alone did not correct these deficits, we explored the potential benefit of HIF-1 gene therapy combined with the intravenous infusion of bone marrow-derived angiogenic cells (BMDACs) cultured with dimethyloxalylglycine (DMOG). DMOG is known to reduce oxidative degradation of HIF-1. The mice treated with a plasmid DNA construct expressing a stabilized mutant form of HIF-1α (CA5-HIF-1α)+BMDACs had more rapid wound closure. By day 17, there were more mice with completely closed wounds in the treated group (χ(2), P=0.05). The dermal blood flow measured by laser Doppler showed significantly increased wound perfusion on day 11. Homing of BMDACs to the burn wound was dramatically enhanced by CA5-HIF-1α gene therapy. HIF-1α mRNA expression in the burn wound was increased after transfection with CA5-HIF-1α plasmid. Our findings offer insight into the pathophysiology of burns in the elderly and point to potential targets for developing new therapeutic strategies.

Skin vasodilation and analgesic effect of a topical nitric oxide-releasing hydrogel

New approaches based on topical treatments are needed for treating pain and impaired dermal blood flow. We used a topical Pluronic F127 hydrogel containing S-nitrosoglutathione (GSNO) as a prodrug to generate free NO, an effector molecule that exerts both dermal vasodilation and antinociceptive effects. GSNO-containing hydrogels underwent gelation above 12°C and released free NO at rates that were directly dependent on the GSNO concentration in the range of 50-150mM. The topical application of this material led to dose-response dermal vasodilation in healthy volunteers and to a reduction of up to 50% of the hypernociception intensity in Wistar rats that were subjected to inflammatory pain. Mechanistic investigations indicated that the antinociceptive effect of the topical F127/GSNO hydrogels is produced by the local activation of the cGMP/PKG/KATP channel-signaling pathway, which was stimulated by the free NO that diffused through the skin. These results expand the scope of the biomedical applications of this material and may represent a new approach for the topical treatment of inflammatory pain.

A New Monitor to Measure Dermal Blood Flow in Critically Ill Patients: A Preliminary Study

Background. Conditions of reduced perfusion are characterized by redistribution of blood flow away from the skin to more vital organs. Objectives. To assess the efficacy of a noninvasive, dermal blood flow (DBF) monitor in detecting changes in perfusion in critically ill patients. Methods. Eleven adult, critically ill patients in a general ICU were studied. DBF, finger plethysmography, and invasive mean arterial pressure (MAP) were recorded over an 8-hour period. DBF was measured using the DermaFlow DBF monitor via a skin probe placed on the anterior chest wall. Sensitivity was evaluated by visual inspection during active states, either induced, for example, fluid administration, or spontaneous, for example, altered hemodynamics, while specificity was evaluated during stable states. Data are expressed in terms of standard deviation of the difference (SDD) between the MAP and each of the tested methods. Results. The DBF detected all true changes detected by MAP while plethysmography detected fewer of these events. Based on SDD, the specificity of the DBF was found to be better than that of plethysmography and close in value to the MAP. Conclusions. This preliminary study suggests that the DBF monitor may be a useful noninvasive method for detecting changes in perfusion in critically ill patients.

Mast cell dependent vascular changes associated with an acute response to cold immersion in primary contact urticaria.

BackgroundWhile a number of the consequences of mast cell degranulation within tissues have been documented including tissue-specific changes such as bronchospasm and the subsequent cellular infiltrate, there is little known about the immediate effects of mast cell degranulation on the associated vasculature, critical to understanding the evolution of mast cell dependent inflammation.ObjectiveTo characterize the microcirculatory events that follow mast cell degranulation.Methodology/Principal FindingsPerturbations in dermal blood flow, temperature and skin color were analyzed using laser-speckle contrast imaging, infrared and polarized-light colorimetry following cold-hand immersion (CHI) challenge in patients with cold-induced urticaria compared to the response in healthy controls. Evidence for mast cell degranulation was established by documentation of serum histamine levels and the localized release of tryptase in post-challenge urticarial biopsies. Laser-speckle contrast imaging quantified the attenuated response to cold challenge in patients on cetirizine. We found that the histamine-associated vascular response accompanying mast cell degranulation is rapid and extensive. At the tissue level, it is characterized by a uniform pattern of increased blood flow, thermal warming, vasodilation, and recruitment of collateral circulation. These vascular responses are modified by the administration of an antihistamine.Conclusions/SignificanceMonitoring the hemodynamic responses within tissues that are associated with mast cell degranulation provides additional insight into the evolution of the acute inflammatory response and offers a unique approach to assess the effectiveness of treatment intervention.

Capsaicin-induced CGRP-mediated vasodilatation of the human skin: influence of gender, female hormones and migraine

Migraine is much more common in women than in men. It is associated with changes in female sex hormone levels with peaks of migraine frequency occurring when estrogen levels drop. Calcitonin gene-related peptide (CGRP) is a potent vasodilating neuropeptide with a pivotal role in migraine headache. Endogenous release of CGRP is induced by capsaicin through activation of the transient receptor potential vanilloid 1 (TRPV1) channel. This study aimed to investigate the influence of gender, hormonal changes and migraine on dermal blood flow (DBF) resulting from capsaicin-induced release of CGRP. Healthy, non-smoking female volunteers (n=16) not using hormonal contraceptives, were investigated weekly during 2 menstrual cycles. Weekly, two doses of capsaicin (300 and 1000µg) and vehicle were applied topically on the skin of both forearms. DBF was assessed before and at 10, 20, 30 and 40 minutes after capsaicin/vehicle application using laser Doppler imaging. DBF is expressed as percentage increase versus baseline and presented as area-under-the-curve from 0 to 40 minutes (AUC, %.min, mean±SEM). Period differences in capsaicin-induced DBF were observed after both doses of capsaicin (p<0.001, repeated-measures ANOVA). During menstruation, capsaicin-induced DBF, expressed as AUC, was larger after 300µg (1488±178 versus 1228±157 %.min, p=0.019; paired T-test) and 1000µg of capsaicin (1639±150 versus 1394±163 %.min, p=0.014) compared to the second week of the secretory phase of the menstrual cycle. Analyses of gender differences and migraine patients versus healthy subjects are ongoing and will be presented at the meeting.

Influence of varying estrogen levels on trigeminal CGRP release in healthy women

Migraine is 2-3 times more prevalent in women than in men, with frequent perimenstrual attacks. TRPV1 receptors on sensory nerve endings of the trigeminal track are important in mediating migraine attacks by releasing the vasodilator calcitonin gene-related peptide (CGRP). Variation in estrogen levels during the menstrual cycle may have an influence on the sensitivity of the TRPV1 receptor or on the amount of CGRP in perivascular nerve terminals and hence on CGRP release. Capsaicin, the active ingredient of hot chili peppers, stimulates the TRPV1 receptor and causes CGRP-dependent vasodilatation [1]. We set up a model to study trigeminal CGRP release in humans. We compared the vasodilator effects of capsaicin application and electrical stimulation on the forehead skin. Healthy women, not using hormonal contraceptives (age: 18-45, n=14), were studied with a Laser Doppler Imager on day 19-21 of their menstrual cycle and on day 1-2 of their menstruation. A 0.2 mM and a 20 mM capsaicin solution were applied to the skin. In addition, iontophoresis of saline was performed as a TRPV1-independent stimulus. Increases in dermal blood-flow (DBF) were measured. Blood samples were collected to measure estrogen levels. We measured higher DBF responses to application of 0.2 mM capsaicin (Max:226±34 a.u.) and 20 mM capsaicin (Max:507±39 a.u.) during day 1-2 (low estrogen levels: 15±2 pg/ml) of the menstruation, than during day 19-21 (high estrogen levels: 67±9 pg/ml) of the menstrual cycle (Max:176±34 a.u. and 432±33 a.u. for 0.2 mM and 20 mM, respectively, P<0.05). There was no difference in DBF responses to electrical stimulation of the forehead skin, suggesting that the observed changes are related to the sensitivity of the TRPV1 receptor. Our results indicate an influence of variation in estrogen levels on trigeminal CGRP release, with the highest reactivity observed around the menstruation when estrogen levels are low. This mechanism may, at least partly, explain the high incidence of migraine attacks during the perimenstrual period.

Switchable skin window induced by optical clearing method for dermal blood flow imaging

Optical imaging techniques have shown tremendous potential for assessing cutaneous microcirculation, but the imaging depth and contrast is limited by the strong scattering of skin. Current skin windows have to be fulfilled by surgical operation and suffer from some side effects. In this study, a switchable skin window was developed by topical application of an optical clearing agent (OCA) and saline on rat skin in vivo. The validity of the skin window was evaluated by the laser speckle contrast imaging technique, and the safety of OCA to the body was tested through histologic examinations. The results indicated that administration of OCA or saline on rat skin in vivo can open or close the window of skin repeatedly for three days. With the repair effect of hyaluronic acid and Vaseline, it is able to repeatedly visualize the dermal blood vessels and flow distribution. Long-term observation shows that there is no abnormal reflection in micro-structure, body weight, organ coefficients, histopathologic lesions, or toxic reactions compared with a control group. This switchable window will provide an effective tool not only for cutaneous microcirculation with laser speckle contrast imaging, but also for diagnosis and treatment of peripheral vascular diseases, including tumor research with various optical imaging techniques.

Nanotechnology Allied to Nitric Oxide Release Materials for Dermatological Applications

It is known that nitric oxide (NO) exerts numerous physiological activities in mammals and it is considered a key mediator in many skin disorders. NO is synthesized in human skin cells where it regulates diverse physiological mechanisms such as dermal blood flow, wound healing, skin pigmentation, and aging. The great challenge of NO-based therapies is to preserve the biological activity of this ephemeral free radical. To this end, new biomaterials have been emerging as platforms for NO release for diverse biomedical applications. This review will discuss the new strategies for releasing/generating controlled amounts of NO for several dermatological applications through the use of nanotechnology. Keywords: Nitric oxide, nanoparticles, skin, wound healing, antibacterial, topical applications

Implications for burn shock resuscitation of a new in vivo human vascular microdosing technique (microdialysis) for dermal administration of noradrenaline

IntroductionSkin has a large dynamic capacity for alterations in blood flow, and is therefore often used for recruitment of blood during states of hypoperfusion such as during burn shock resuscitation. However, little is known about the blood flow and metabolic consequences seen in the dermis secondary to the use vasoactive drugs (i.e. noradrenaline) for circulatory support. The aims of this study were therefore: to develop an in vivo, human microdosing model based on dermal microdialysis; and in this model to investigate effects on blood flow and metabolism by local application of noradrenaline and nitroglycerin by the microdialysis system simulating drug induced circulatory support. MethodNine healthy volunteers had microdialysis catheters placed intradermally in the volar surface of the lower arm. The catheters were perfused with noradrenaline 3 or 30mmol/L and after an equilibrium period all catheters were perfused with nitroglycerine (2.2mmol/L). Dermal blood flow was measured by the urea clearance technique and by laser Doppler imaging. Simultaneously changes in dermal glucose, lactate, and pyruvate concentrations were recorded. ResultsNoradrenaline and nitroglycerine delivered to the dermis by the microdialysis probes induced large time- and dose-dependent changes in all variables. We particularly noted that tissue glucose concentrations responded rapidly to hypoperfusion but remained higher than zero. Furthermore, vasoconstriction remained after the noradrenaline administration implicating vasospasm and an attenuated dermal autoregulatory capacity. The changes in glucose and lactate by vasoconstriction (noradrenaline) remained until vasodilatation was actively induced by nitroglycerine. ConclusionThese findings, i.e., compromised dermal blood flow and metabolism are particularly interesting from the burn shock resuscitation perspective where noradrenaline is commonly used for circulatory support. The importance and clinical value of the results obtained in this in vivo dermal model in healthy volunteers needs to be further explored in burn-injured patients.

Increased stability of S-nitrosothiol solutions via pH modulations

S-nitrosothiol (RSNO) solutions represent a valuable source of nitric oxide and could be used as topical vasodilators, but their fast decomposition rate poses a serious obstacle to their potentially widespread therapeutic use. Our aim was to characterize and quantify the effect of pH on S-nitrosothiol formation and decomposition in simple aqueous solutions of S-nitrosoglutathione (GSNO), S-nitroso-N-acetylcysteine (SNAC) and S-nitroso-3-mercaptopropionic acid (SN3MPA). Furthermore, we investigated the effect of storage pH on the stability of GSNO incorporated in poly(ethylene glycol)/ poly(vinyl alcohol) matrices. S-nitrosothiol concentrations were measured spectrophotometrically and laser Doppler scanning method was used to assess dermal blood flow. GSH and NAC solutions reached a complete transformation to nitrosothiols when synthesized using acidic NaNO2 solution. The initial concentration of all investigated RSNOs decreased more slowly with pH adjusted to mildly basic values (8.4–8.8) for the storage period. Polymer gels of PVA/PEG compositions at mildly basic storage pH further reduced the decomposition rate succeeding to contain 46.8% of the initial GSNO concentration for 25 days. This amount of topically administered GSNO was still capable of increasing the dermal blood flow over 200% in human subjects.

Burn wound healing time assessed by laser Doppler imaging (LDI). Part 1: Derivation of a dedicated colour code for image interpretation

IntroductionLaser Doppler imaging produces a colour-coded image of dermal blood flow, which can be used to quantify the inflammatory response in a burn. The original colour palette had arbitrary boundaries, which inexperienced clinicians found difficult to interpret. The aim of this study was to define clinically useful boundaries that would assist in the prediction of burn healing potential. MethodWe conducted a prospective, multi-centre study of burns in adults and children. LDI scans were performed between 48h and 5 days after injury. The burns were assessed clinically and photographed on day of scan, day 14 and day 21 post-injury. Areas healed at day 14, healed between day 14 and 21 and unhealed at day 21 were identified on the LDI scan. The flow values for the pixels in these regions were analysed to calculate boundaries between the three healing categories. ResultsWe recruited 137 patients (ages 1–88 years, 65% male); 392 LDI scans contained 433 different burn sites; 109 regions of interest were studied. Analysis allowed us to define ranges for the three healing categories: HP14 colour coded red, >600PU; HP14–21, yellow, 260–440PU; HP>21, blue, <200PU; separated by two overlap regions pink, 440–600PU and green, 200–260PU. Blue was subdivided to show the very high association between LDI<140PU and non-healing at day 21. ConclusionWe have devised a new colour palette for LDI burn imaging based on healing times of a series of burns. Validation of this palette is described separately, in Part 2.

Non-invasive imaging of cold-induced pain related dermal blood flow

Non-invasive imaging of cold-induced pain related dermal blood flow