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Cell regenerative therapy is a modern solution for difficult-to-heal wounds. Keratinocytes, the most common cell type in the skin, are difficult to obtain without the creation of another wound. Stem cell differentiation towards keratinocytes is a challenging process, and it is difficult to reproduce in chemically defined media. Nevertheless, a co-culture of keratinocytes with stem cells usually achieves efficient differentiation. This systematic review aims to identify the secretions of normal human keratinocytes reported in the literature and correlate them with the differentiation process. An online search revealed 338 references, of which 100 met the selection criteria. A total of 80 different keratinocyte secretions were reported, which can be grouped mainly into cytokines, growth factors, and antimicrobial peptides. The growth-factor group mostly affects stem cell differentiation into keratinocytes, especially epidermal growth factor and members of the transforming growth factor family. Nevertheless, the reported secretions reflected the nature of the involved studies, as most of them focused on keratinocyte interaction with inflammation. This review highlights the secretory function of keratinocytes, as well as the need for intense investigation to characterize these secretions and evaluate their regenerative capacities.

Transdermal iontophoresis offers an in vivo alternative to the strain-gauge model for measurement of vascular function but is limited due to lack of technical solutions for outcome assessment. The aims of this study were to, after measurement by polarized reflectance spectroscopy (PRS), use pharmacodynamic dose-response analysis on responses to different concentrations of acetylcholine (ACh); and to examine the effect of three consecutively administered iontophoretic current pulses. The vascular responses in 15 healthy volunteers to iontophorised ACh (5 concentrations, range 0.0001% to 1%, three consecutive pulses of 0.02 mA for 10 min each) were recorded using PRS. Data were fitted to a four-parameter logistic dose response model and compared. Vascular responses were quantifiable by PRS. Similar pharmacodynamic dose response curves could be generated irrespectively of the ACh concentration. Linearly increasing maximum vasodilatory responses were registered with increasing concentration of ACh. A limited linear dose effect of the concentration of ACh was seen between pulses. Polarized reflectance spectroscopy is well suited for measuring vascular responses to iontophoretically administrated ACh. The results of this study support further development of iontophoresis as a method to study vascular function and pharmacological responses in vivo., (© 2022. The Author(s).)

The rules governing Medicinal Products in the European Union necessitates the production of cell-based therapy in good manufacturing practice facilities. The produced cells may need several hours in transportation to reach the application sites. In this study, we investigated four candidate solutions for transporting human keratinocytes. The solutions are (1) normal saline, (2) saline with 2.5% human serum albumin (Saline + HSA), (3) chemically defined, xeno-free keratinocyte media and (4) keratinocyte media with pituitary bovine extract (PBE-media). One million keratinocytes from three donors were suspended in each solution and kept at 4 °C for up to 24 h. Cells kept in Saline + HSA showed higher viability after 1, 3 and 24 h. Then, equal number of viable cells were seeded on collagenous matrix and cultured for 48 h. The adhesion and colonization were higher in the cells kept in PBE-media, while the keratinocyte surface marker, cytokeratin 14, was present in all studied groups. These results confirmed the suitability of Saline + HSA as a cell transportation solution for clinical use, which will be the choice for the planned clinical trial. Keratinocyte PBE-media can be an alternative for cells transported for research purpose, if the same media type is going to be used in the following experiments.

Objectives: Measurement of perfusion is an established method to evaluate the depth of burns. However, high accuracy is only achievable >48 h after injury. The aim of the study was to investigate if measurement of blood flow pulsatility, combined with perfusion measurement, can improve early assessment of burn depth using laser speckle contrast imaging (LSCI)., Methods: Perfusion and pulsatility were measured with LSCI in 187 regions of interest in 32 patients, between 0 and 5 days after injury. The reproducibility of pulsatility was tested for recording durations between 1 and 12 s. The most reproducible duration was chosen, and receiver operator characteristics were created to find suitable pulsatility cut-offs to predict surgical need., Results: A measurement duration of 8 s resulted in a good reproducibility of the pulsatility (%CV: 15.9%). Longer measurement durations resulted in a small improvement of the accuracy of the assessment. A pulsatility of <1.45 (Perfusion Units) 2 on day 0-2 after injury predicted surgical need with a sensitivity of 100% (95% CI: 83.2-100%), specificity of 100% (95% CI: 95.2-100%), a positive predictive value of 100%, and a negative predictive value of 100%. Pulsatility was not significantly different when comparing measurements done day 0-2 to day 3-5. Perfusion was however significantly higher day 3-5 compared to day 0-2 for wounds healing within 3 weeks., Conclusion: Measurement of pulsatility improves the accuracy of the assessment of burns with LSCI and makes it possible to predict the need for surgery during day 0-2 after injury with a high accuracy., (Copyright © 2020 Elsevier Ltd and ISBI. All rights reserved.)

We present in this paper the application of deep convolutional neural networks (CNNs), which is a state-of-the-art artificial intelligence (AI) approach in machine learning, for automated time-independent prediction of burn depth. Color images of four types of burn depth injured in first few days, including normal skin and background, acquired by a TiVi camera were trained and tested with four pretrained deep CNNs: VGG-16, GoogleNet, ResNet-50, and ResNet-101. In the end, the best 10-fold cross-validation results obtained from ResNet-101 with an average, minimum, and maximum accuracy are 81.66, 72.06, and 88.06%, respectively; and the average accuracy, sensitivity, and specificity for the four different types of burn depth are 90.54, 74.35, and 94.25%, respectively. The accuracy was compared with the clinical diagnosis obtained after the wound had healed. Hence, application of AI is very promising for prediction of burn depth and, therefore, can be a useful tool to help in guiding clinical decision and initial treatment of burn wounds., (© American Burn Association 2019. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Objectives: Laser speckle contrast imaging (LSCI) is an emerging technique for the assessment of burns in humans and interobserver differences have not been studied. The aim of this study was to compare assessments of perfusion images by different professional groups regarding (i) perfusion values and (ii) burn depth assessment., Methods: Twelve observers without LSCI experience were included. The observers were evenly recruited from three professional groups: plastic surgeons with experience in assessing burns, nurses with experience in treating burns, and junior doctors with limited experience of burns. Ten cases were included. Each case consisted of one digital photo of the burn with a pre-marked region of interest (ROI) and two unmarked perfusion images of the same area. The first and the second perfusion image was from 24h and 72-96h after injury, respectively. The perfusion values from both perfusion images were used to generate a LSCI recommendation based on the perfusion trend (the derivative between the two perfusion values). As a last step, each observer was asked to estimate the burn depth using their clinical experience and all available information. Intraclass correlation (ICC) was calculated between the different professional groups and among all observers., Results: Perfusion values and perfusion trends between all observers had an ICC of 0.96 (95% CI 0.91-0.99). Burn depth assessment by all observers yielded an ICC of 0.53 (95% CI: 0.31-0.80) and an accuracy of 0.53 (weighted kappa). LSCI recommendations generated by all observers had an ICC of 0.95 (95% CI: 0.90-0.99)., Conclusion: Observers can reliably identify the same ROI, which results in observer-independent perfusion measurements, irrespective of burn experience. Extensive burn experience did not further improve burn depth assessment. The LSCI recommendation was more accurate in all professional groups. Introducing LSCI measurements would be likely improve early assessment of burns., (Copyright © 2019 Elsevier Ltd and ISBI. All rights reserved.)

Severe burns are often treated by means of autologous skin grafts, preferably following early excision of the burnt tissue. In the case of, for example, a large surface trauma, autologous skin cells can be expanded in vitro prior to transplantation to facilitate the treatment when insufficient uninjured skin is a limitation. In this study we have analyzed the impact of the enzyme (trypsin or accutase) used for cell dissociation and the incubation time on cell viability and expansion potential, as well as expression of cell surface markers indicative of stemness. Skin was collected from five individuals undergoing abdominal reduction surgery and the epidermal compartment was digested in either trypsin or accutase. Trypsin generally generated more cells than accutase and with higher viability; however, after 7 days of subsequent culture, accutase-digested samples tended to have a higher cell count than trypsin, although the differences were not significant. No significant difference was found between the enzymes in median fluorescence intensity of the analyzed stem cell markers; however, accutase digestion generated significantly higher levels of CD117- and CD49f-positive cells, but only in the 5 h digestion group. In conclusion, digestion time appeared to affect the isolated cells more than the choice of enzyme.

Background: Changes in microvascular perfusion in scalds in children during the first four days, measured with laser speckle contrast imaging (LSCI), are related to the time to healing and need for surgical intervention. The aim of this study was to determine the accuracy (sensitivity and specificity) of LSCI on different days after injury in the prediction of healing outcome and if the accuracy can be improved by combining an early and a late measurement. Also, the accuracy of LSCI was compared with that of clinical assessment., Methods: Perfusion was measured between 0-24h and between 72-96h using LSCI in 45 children with scalds. On the same occasions, burn surgeons assessed the burns as healing <14days or healing >14days/surgery. Receiver operating characteristic (ROC) curves were constructed for the early and late measurement and for the double measurement (DM) using two different methods., Results: Sensitivity and specificity were 92.3% (95% CI: 64.0-99.8%) and 78.3% (95% CI: 69.9-85.3%) between 0-24h, 100% (95% CI: 84.6-100%) and 90.4% (95% CI: 83.8-94.9%) between 72-96h, and was 100% (95% CI: 59.0-100%) and 100% (95% CI: 95.1-100%) when combining the two measurements into a modified perfusion trend. Clinical assessment had an accuracy of 67%, Cohen's κ=0.23., Conclusion: The perfusion in scalds between 72-96h after injury, as measured using LSCI, is highly predictive of healing outcome in scalds when measured. The predictive value can be further improved by incorporating an early perfusion measurement within 24h after injury., (Copyright © 2017 Elsevier Ltd and ISBI. All rights reserved.)

Forearm skin hyperemia during release after brachial occlusion has been proposed for evaluating peripheral arterial disease and endothelial dysfunction. We used a novel fiberoptic system integrating Laser Doppler Flowmetry and Diffuse Reflectance Spectroscopy for a comprehensive pointwise model based microcirculation characterization. The aim was to evaluate and compare the temporal and the spatiotemporal variabilities in forearm skin microcirculation parameters (speed resolved perfusion; low speed <1mm/s, Perf SR, <1 ; mid-speed 1-10mm/s, high speed >10mm/s, and total perfusion (Perf SR, tot ); the concentration and oxygenation of red blood cells, C RBC and S O2 ). Ten healthy subjects underwent arterial and venous forearm occlusions (AO, VO), repeated within one week. The repeatability was calculated as the coefficient of variation (CV) and the agreement as the intra-class correlation coefficient (ICC). The temporal CVs for conventional perfusion, Perf conv , Perf SR, tot , C RBC and S O2 were 14%, 12%, 9% and 9%, respectively, while the ICC were >0.75 (excellent). The perfusion measures generally had a higher spatiotemporal than temporal variability, which was not the case for S O2 and C RBC . The corresponding spatiotemporal CVs were 33%, 32%, 18% and 15%, respectively. During VO, C RBC had a CV<35% and ICC>0.40 (fair-good), and after release this was the case for C RBC (AO and VO), S O2 (VO) and Perf SR, <1 (VO). In conclusion, the skin microcirculation parameters showed excellent temporal repeatability, while the spatiotemporal repeatability especially for perfusion was poorer. The parameters with acceptable repeatability and fair-good agreement were: C RBC during and after release of VO, the Perf SR, <1 after release of VO, the S O2 and the C RBC after release of AO. However, the value of these parameters in discriminating endothelial function remains to be studied., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Objective: Insulin causes capillary recruitment in muscle and adipose tissue, but the metabolic and microvascular effects of insulin in the skin have not been studied in detail. The aim of this study was to measure glucose metabolism and microvascular blood flow in the skin during local insulin delivery and after an oral glucose load., Methods: Microdialysis catheters were inserted intracutanously in human subjects. In eight subjects two microdialysis catheters were inserted, one perfused with insulin and one with control solution. First the local effects of insulin was studied, followed by a systemic provocation by an oral glucose load. Additionally, as control experiment, six subjects did not recieve local delivery of insulin or the oral glucose load. During microdialysis the local blood flow was measured by urea clearance and by laser speckle contrast imaging (LSCI)., Results: Within 15 minutes of local insulin delivery, microvascular blood flow in the skin increased (urea clearance: P=.047, LSCI: P=.002) paralleled by increases in pyruvate (P=.01) and lactate (P=.04), indicating an increase in glucose uptake. An oral glucose load increased urea clearance from the catheters, indicating an increase in skin perfusion, although no perfusion changes were detected with LSCI. The concentration of glucose, pyruvate and lactate increased in the skin after the oral glucose load., Conclusion: Insulin has metabolic and vasodilatory effects in the skin both when given locally and after systemic delivery through an oral glucose load., (© 2016 John Wiley & Sons Ltd.)

Background: Microvascular perfusion changes in scalds in children during the first weeks after injury is related to the outcome of healing, and measurements of perfusion, based on laser Doppler imaging, have been used successfully to predict the need for excision and grafting. However, the day-to-day changes in perfusion during the first weeks after injury have not to our knowledge been studied in detail. The aim of this study, based on a conservative treatment model where excision and grafting decisions were delayed to day 14 after injury, was to measure changes in perfusion in scalds using laser speckle contrast imaging (LSCI) during the first three weeks after injury., Methods: We measured perfusion with LSCI in 34 patients at regular intervals between 6h after injury until complete reepithelialization or surgery. Duration of healing was defined as the time to complete reepithelialization., Results: Less perfusion, between 6 and 96h after injury, was associated with longer duration of healing with the strongest association occurring between 72 and 96h. Burns that healed within 14 days had relatively high initial perfusion, followed by a peak and subsequent slow decrease. Both the maximum perfusion and the time-to-peak were dependent on the severity of the burn. Burns that needed excision and grafting had less initial perfusion and a gradual reduction over time., Conclusion: The perfusion in scalds in children shows characteristic patterns during the first weeks after injury depending on the duration of wound healing, the greatest difference between wounds of different severity being on the 4th day. Perfusion should therefore preferably be measured on the fourth day if it is to be used in the assessment of burn depth., (Copyright © 2015 Elsevier Ltd and ISBI. All rights reserved.)

Background: Venous occlusion in the skin is difficult to detect by existing measurement techniques. Our aim was to find out whether Tissue Viability Imaging (TiVi) was better at detecting venous occlusion by comparing it with results of laser Doppler flowmetry (LDF) during graded arterial and venous stasis in human forearm skin., Methods: Arterial and venous occlusions were simulated in 10 healthy volunteers by inflating a blood pressure cuff around the upper right arm. Changes in the concentration of red blood cells (RBC) were measured using TiVi, while skin perfusion and concentration of moving red blood cells (CMBC) were measured using static indices of LDF during exsanguination and subsequent arterial occlusion, postocclusive reactive hyperaemia, and graded increasing and decreasing venous stasis., Results: During arterial occlusion there was a significant reduction in the mean concentration of RBC from baseline, as well as in perfusion and CMBC (p<0.008). Venous occlusion resulted in a significant 28% increase in the concentration of RBC (p=0.002), but no significant change in perfusion (mean change -14%) while CMBC decreased significantly by 24% (p=0.02). With stepwise increasing occlusion pressures there was a significant rise in the TiVi index and reduction in perfusion (p=0.008), while the reverse was seen when venous flow was gradually restored., Conclusion: The concentration of RBC measured with TiVi changes rapidly and consistently during both total and partial arterial and venous occlusions, while the changes in perfusion, measured by LDF, were less consistent. This suggests that TiVi could be a more useful, non-invasive clinical monitoring tool for detecting venous stasis in the skin than LDF., (Copyright © 2015. Published by Elsevier Inc.)

Introduction: Microvascular changes in the skin due to pharmacological and physiological provocations can be used as a marker for vascular function. While laser Doppler flowmetry (LDF) has been used extensively for measurement of skin microvascular responses, Laser Speckle Contrast Imaging (LSCI) and Tissue Viability Imaging (TiVi) are novel imaging techniques. TiVi measures red blood cell concentration, while LDF and LSCI measure perfusion. Therefore, the aim of this study was to compare responses to provocations in the skin using these different techniques., Method: Changes in skin microcirculation were measured in healthy subjects during (1) iontophoresis of sodium nitroprusside (SNP) and noradrenaline (NA), (2) local heating and (3) post-occlusive reactive hyperemia (PORH) using LDF, LSCI and TiVi., Results: Iontophoresis of SNP increased perfusion (LSCI: baseline 40.9±6.2 PU; 10-min 100±25 PU; p<0.001) and RBC concentration (TiVi: baseline 119±18; 10-min 150±41 AU; p = 0.011). No change in perfusion (LSCI) was observed after iontophoresis of NA (baseline 38.0±4.4 PU; 10-min 38.9±5.0 PU; p = 0.64), while RBC concentration decreased (TiVi: baseline 59.6±11.8 AU; 10-min 54.4±13.3 AU; p = 0.021). Local heating increased perfusion (LDF: baseline 8.8±3.6 PU; max 112±55 PU; p<0.001, LSCI: baseline 50.8±8.0 PU; max 151±22 PU; p<0.001) and RBC concentration (TiVi: baseline 49.2±32.9 AU; max 99.3±28.3 AU; p<0.001). After 5 minutes of forearm occlusion with prior exsanguination, a decrease was seen in perfusion (LDF: p = 0.027; LSCI: p<0.001) and in RBC concentration (p = 0.045). Only LSCI showed a significant decrease in perfusion after 5 minutes of occlusion without prior exsanguination (p<0.001). Coefficients of variation were lower for LSCI and TiVi compared to LDF for most responses., Conclusion: LSCI is more sensitive than TiVi for measuring microvascular changes during SNP-induced vasodilatation and forearm occlusion. TiVi is more sensitive to noradrenaline-induced vasoconstriction. LSCI and TiVi show lower inter-subject variability than LDF. These findings are important to consider when choosing measurement techniques for studying skin microvascular responses.

Objective: After iontophoresis of vasoactive drugs into the skin, a decrease in perfusion is commonly observed. We delivered vaso-active drugs by iontophoresis using different delivery protocols to study how these affect this decrease in perfusion as measured using LDF., Methods: We measured skin perfusion during iontophoresis of (ACh), MCh, and NA using a single pulse or separate pulses at different skin sites, and during repeated delivery of ACh at the same site., Results: Perfusion half-life was 6.1 (5.6-6.6) minutes for ACh and 41 (29-69) minutes for MCh (p < 0.001). The maximum response with multiple pulses of ACh iontophoresis was lower than with a single pulse, 30 (22-37) PU vs. 43 (36-50) PU, p < 0.001. Vasoconstriction to NA was more rapid with a single pulse than with multiple pulses. The perfusion half-life of ACh decreased with repeated delivery of ACh at the same site-first 16 (14-18), second 5.9 (5.1-6-9) and third 3.2 (2.9-3.5) minutes, p < 0.001., Conclusions: The drug delivery protocol affects microvascular responses to iontophoresis, possibly as a result of differences in the dynamics of local drug concentrations. Perfusion half-life may be used as a measure to quantify the rate of perfusion recovery after iontophoresis of vasoactive drugs., (© 2015 John Wiley & Sons Ltd.)

Objective: Insulin has direct effects on blood flow in various tissues, most likely due to endothelial NO production. We investigated whether insulin delivered to the skin by iontophoresis increases microvascular perfusion and whether this effect is partly or completely mediated by the release of NO., Methods: In healthy subjects, regular insulin and monomeric insulin were delivered to the skin by cathodal iontophoresis. The skin was pretreated either with L-NAME or control solution (PBS) using anodal iontophoresis. Microvascular responses were measured using laser Doppler flowmetry., Results: A dose-dependent increase in perfusion was observed during iontophoresis of regular and monomeric insulin. The maximum perfusion was significantly elevated compared with control after PBS (regular insulin 53.6 (12.7-95.6) PU vs. 4.2 (3.4-4.8) PU, p = 0.002; monomeric insulin 32.6 (8.9-92.6) PU vs. 5.9 (3.4-56.0) PU, p = 0.03). The microvascular response to insulin was abolished after L-NAME (regular insulin: 25.6 (11.6-54.4) PU vs. control: 4.7 (2.9-11.5) PU, p = 0.15; monomeric insulin 10.9 (5.4-56.8) PU vs. control: 4.7 (2.9-11.5) PU, p = 0.22)., Conclusions: The main finding is that iontophoresis of insulin induces a dose-dependent vasodilation in the skin, which could be suppressed after pretreatment with a NO synthase inhibitor. This suggests that vasodilation in the skin after iontophoresis of insulin is mediated by the NO pathway., (© 2013 John Wiley & Sons Ltd.)

Objective: To evaluate whether TiVi, a technique based on polarized light, could measure the change in RBC concentration during local heating in healthy volunteers., Methods: Using a custom-made transparent heater, forearm skin was heated to 42 °C for 40 minutes while the change in RBC concentration was measured with TiVi. The perfusion response during local heating was measured at the same time with Laser Doppler flowmetry., Results: Mean RBC concentration increased (91 ± 34 vs. 51 ± 34 A.U. at baseline, p < 0.001). The spatial heterogeneity of the RBC concentration in the measured skin areas was 26 ± 6.4% at baseline, and 23 ± 4.6% after 40 minutes of heating. The mean RBC concentrations in two skin sites were highly correlated (0.98 at baseline and 0.96 after 40 minutes of heating). The change in RBC concentration was less than the change in perfusion, measured with LDF. Unlike with LDF, a neurally mediated peak was not observed with TiVi in most of the test subjects., Conclusions: TiVi is a valuable technique for measuring the microvascular response to local heating in the skin, and offers a high reproducibility for simultaneous measurements at different skin sites, provided carefully controlled experiments are ensured., (© 2012 John Wiley & Sons Ltd.)

Objective: Microdialysis enables drug delivery in the skin and simultaneous measurement of their effects. The present study aimed to evaluate dose-dependent changes in blood flow and metabolism during microdialysis of norepinephrine and vasopressin., Methods: We investigated whether increasing concentrations of norepinephrine (NE, 1.8-59 μmol/L) and vasopressin (VP, 1-100 nmol/L), delivered sequentially in one catheter or simultaneously through four catheters, yield dose-dependent changes in blood flow (as measured using urea clearance) and metabolism (glucose and lactate)., Results: We found a significant dose-dependent vasoconstriction with both drugs. Responses were characterized by a sigmoid dose response model. Urea in the dialysate increased from a baseline of 7.9 ± 1.7 to 10.9 ± 0.9 mmol/L for the highest concentration of NE (p < 0.001) and from 8.1 ± 1.4 to 10.0 ± 1.7 mmol/L for the highest concentration of VP (p = 0.037). Glucose decreased from 2.3 ± 0.7 to 0.41 ± 0.18 mmol/L for NE (p = 0.001) and from 2.7 ± 0.6 to 1.3 ± 0.5 mmol/L for VP (p < 0.001). Lactate increased from 1.1 ± 0.4 to 2.6 ± 0.5 mmol/L for NE (p = 0.005) and from 1.1 ± 0.4 to 2.6 ± 0.5 mmol/L for VP (p = 0.008). There were no significant differences between responses from a single catheter and from those obtained simultaneously using multiple catheters., Conclusions: Microdialysis in the skin, either with a single catheter or using multiple catheters, offers a useful tool for studying dose response effects of vasoactive drugs on local blood flow and metabolism without inducing any systemic effects., (© 2012 John Wiley & Sons Ltd.)

Objective: The aim of this study was to evaluate the urea clearance technique for the measurement of drug-induced blood flow changes in human skin and compare it to two non-invasive techniques: polarization light spectroscopy and laser Doppler perfusion imaging., Methods: Fifteen microdialysis catheters were placed intracutaneously on the volar aspect of the forearms of healthy human subjects and were perfused with nitroglycerine, noradrenaline, and again nitroglycerine to induce local tissue hyperemia, hypoperfusion, and hyperemia, respectively., Results: Urea clearance, but not the other techniques, detected the changes in blood flow during changes in flow. The last hyperemic response was detected by all three methods., Conclusion: Urea clearance can be used as a relatively simple method to estimate blood flow changes during microdialysis of vasoactive substances, in particular when the tissue is preconditioned in order to enhance the contrast between baseline and the responses to the provocations. Our results support that, in the model described, urea clearance was superior to the optical methods as it detected both the increases and decrease in blood flow, and the returns to baseline between these periods., (© 2011 John Wiley & Sons Ltd.)

Assessment of microvascular function is of major importance in understanding the physiology of the vasculature and in investigating the vascular effects of pathological conditions. Transdermal iontophoresis can be used to non-invasively introduce vasoactive drugs into the skin. The response of the local cutaneous microvasculature to these drugs can be measured by methods such as laser Doppler flowmetry. Although the technique has been used for over two decades, there are still important methodological issues to be resolved. This review describes the technique of iontophoresis as well as its development during recent years, while focusing on how iontophoresis can be used as an in-vivo model for studying physiologic mechanisms and on the analysis and interpretation of dose-response data., (2010 Elsevier Inc. All rights reserved.)

When one is studying the physiology of the cutaneous microcirculation there is a need for relevant non-invasive and versatile techniques. In this study we used a new optical device, the tissue viability imager (TiVi), to map changes in cutaneous microvascular concentrations of red blood cells during iontophoresis of vasoactive substances (noradrenaline (NA) and phenylephrine (Phe) for vasoconstriction and acetylcholine (ACh) and sodium nitroprusside (SNP) for vasodilatation). We aimed to present data both individually and pooled, using a four-variable logistic dose response model that is commonly used in similar in vitro vascular studies. The accuracy of the TiVi was also investigated by calculating the coefficient of variation and comparing it with similar tests previously done using laser Doppler imaging. Tests were also performed using the TiVi and LDPI simultaneously to further compare the two methods. Results showed that the TiVi is capable of quantifying vascular responses to iontophorised noradrenaline and phenylephrine without the need to increase background flow first. Fitting the TiVi data to the dose response model resulted in ED(50)-values with narrow confidence intervals and acceptable r(2) values. Mean ED(50)-values for the TiVi did not differ significantly from similar values obtained using laser Doppler. Results further seem to suggest that when the blood perfusion increases during vasodilatation in skin the initial phase relies mainly on an increase in red blood cell concentration whereas the further perfusion increase is due to an increase in red blood cell velocity.

Background/aims: The analysis of blood flow responses to iontophoresis of vasoactive drugs is often limited to evaluation of maximum responses. In this study, a time-response model is proposed for the blood flow responses to vasoactive drugs applied by iontophoresis., Methods: The microvascular bed is represented as a single compartment with a zero-order influx of the drugs from the electrode and a first-order clearance due to diffusion and blood flow. The blood flow response to the local drug dose is described using the E(max) model., Results: The model accurately describes the blood flow responses to acetylcholine and sodium nitroprusside during a single iontophoretic current pulse. There is a significant clearance out of the microvascular bed during iontophoresis which depends on the type of drug administered., Conclusion: The model enables an accurate estimation of response parameters such as ED50 and maximum response, even if the true maximum blood flow is not obtained. The results suggest that due to clearance from the microvascular bed, the local drug dose during a single pulse of current is not linearly proportional to current strength multiplied by pulse duration.

Objectives: To investigate serotonin (5HT) locally in burned and uninjured skin (intracutaneous) by microdialysis, and simultaneously record urinary and blood values in the same subjects. For comparison, serotonin values were also measured in skin of healthy controls., Design and Setting: An experimental study in burned patients with of more than 25% TBSA (total burn surface area) % in an 8-bed tertiary burns unit, serving about 3.5 million persons., Patients and Methods: Six subjects with a median TBSA% of 59% (range 33.5-90), and five healthy controls were examined by intracutaneous microdialysis of the skin., Results: 5HT was increased in burned patients, compared with controls. This increase was tenfold in skin and was noted both in uninjured and burned skin. The highest values were recorded on day 1 (median 16.1nmol in uninjured and 9.5nmol in burned skin) and day 2 (15.6nmol in uninjured and 13.4nmol in burned skin). A rapid reduction was noted on day 3 (4.9nmol in uninjured and 3.8nmol in burned skin). The corresponding value for control subjects was 1.3nmol. The 5HT in blood was twice normal on day 2, and gradually reduced on days 3 and 4 (3189, 3035 and 2573nmol, respectively). Urinary 5HT concentrations were increased only on day 2 at 1755nmol and thereafter returned to the normal range on days 3 and 4 (1248 and 1344nmol, respectively)., Conclusions: We showed that microdialysis may be used in the critical care of burns, and local skin serotonin concentrations examined continuously for several days. The findings of significantly raised tissue serotonin concentrations, compared to that in blood and urine, suggests that serotonin may be important in local vascular control and formation of oedema.

Background/aims: Many clinical conditions that affect the microcirculation of the skin are still diagnosed and followed up by observational methods alone in spite of the fact that non-invasive, more user-independent and objective methods are available today. Limited portability, high cost, lack of robustness and non-specificity of findings are among the factors that have hampered the implementation of these methods in a clinical setting. The aim of this study is to present and evaluate a new, portable and easy-to-use imaging technology for investigation of the red blood cell (RBC) concentration in the skin microvasculature based on the method of polarization light spectroscopy using modified standard digital camera technology., Methods: The use of orthogonal linear polarization filters over both the flash source and the detector array removes the polarization-retaining light reflected from the epidermal layer. Only the depolarized light backscattered from the papillary dermal matrix reaches the detector array. By separating the RGB color planes of an image acquired in this manner and applying a dedicated image processing algorithm, spectroscopic information about the chromophores in the dermal tissue can be attained. If the algorithm is based on a differential principle in which the normalized differences between the individual values of the red and green color plane are calculated, tissue components with similar spectral signature in both planes are suppressed, while components with different spectral signatures such as RBCs are enhanced., Results: In vitro fluid models compare well with theory and computer simulations in describing a linear relationship between the imager output signal termed the tissue viability index (TiVi(index)) and RBC concentration in the physiological range of 0-4% RBC fraction of tissue volume (cc=0.997, n=20). The influence of oxygen saturation on the calculated RBC concentration is limited to within -3.9% for values within the physiological range (70-100% oxygen saturation). Monte Carlo simulations provide information about the sampling depth (about 0.5 mm on the average) of the imaging system. In vivo system evaluation based on iontophoresis of acetylcholine displays a heterogeneous pattern of vasodilatation appearing inside the electrode area after about 10 min. Topical application of methyl nicotinate and clobetasol propionate further demonstrates the capacity to document the extent and intensity of both an increase (erythema) and a decrease (blanching) in the skin RBC concentration without movement artifact and with compensation for irregularity in pigmentation., Conclusions: Polarization light spectroscopy imaging for assessment of RBC concentration in the skin microvasculature is a robust and accessible technique for the clinical setting. Additionally, the technique has pre-clinical research applications for investigation of the spatial and temporal aspects of skin erythema and blanching as well as a potential role in drug development, skin care product development and skin toxicological assessment.

Frequent unloading is vital to avoid pressure ulcers of the seat area in patients with injuries to the spinal cord. The duration of unloading is probably as important as that of the sitting period in the prophylaxis of pressure ulcers. The aim of this study was to investigate the microcirculatory reactions after occlusion of the buttock skin after repeated ischaemic provocation. The perfusion of buttock skin was studied with a laser Doppler perfusion imager (LDPI) in healthy people after short and long periods of sitting (repeated four times). The perfusion increased significantly during the consecutive loadings compared with the first loading, and this effect was more profound after the long load. Repeated periods of ischaemia of the buttock skin without allowing the tissues to recover resulted in increasing reactive hyperaemia, and are therefore probably more damaging than single loadings. This is important when establishing clinical guidelines for the prophylaxis of pressure ulcers in patients with spinal cord injuries.

Non blanchable erythema, i.e. stage I pressure ulcer, is common in patients in acute and geriatric care and in nursing homes. Research has shown that this type of lesions is prone to develop into more severe pressure ulcers. The peripheral skin blood perfusion is of major importance for the development of pressure ulcers. The aim of this study was to explore the peripheral skin blood perfusion over time, in areas with non blanchable erythema and in corresponding undamaged areas on the opposite side of the body. A total of 19 measurements were performed, over time, using a laser Doppler perfusion imager. The blood flow distribution profiles over areas with non blanchable erythema and undamaged skin were found to be different. As the area of the non blanchable erythema decreased, the blood perfusion distribution profiles gradually became more heterogeneous; an area of high blood perfusion in the centre of the lesions was seen and the perfusion successively decreased closer to the edge. These results indicate that there are differences in blood perfusion between skin areas of non blanchable erythema and undamaged skin. The results also indicate that the visible redness in areas with non blanchable erythema is related to altered blood perfusion. The skin blood perfusion also seems to increase in relation to the size of the non blanchable erythema.

In patients with spinal cord injuries (n=8) and healthy controls (n=8) the hyperaemic response in the buttock skin after sitting on a hard surface was studied using a laser Doppler perfusion imager. They sat for three minutes (short load), or 15 minutes (long load). An exponential mathematical function was used to compare the mean perfusion during the observed interval. The results showed that preloading perfusion is significantly higher among patients than healthy subjects. In both groups, the microcirculation of the skin increased significantly after loading, and peak perfusion was significantly lower after the short load. The mean perfusion was higher among the patients after both loadings, which suggests that there was stronger ischaemic provocation. The main outcome was that there was a dose-response relation between duration of loading and intensity of reactive hyperaemia, and that patients with spinal cord injuries have greater perfusion before and after loading than healthy controls.

Introduction: Established fluid treatment formulas for burn injuries have been challenged as studies have shown the presence of tissue hypoxia during standard resuscitation. Such findings suggest monitoring at the tissue level. This study was performed in patients with major burn injuries to evaluate the microdialysis technique for the continuous assessment of skin metabolic changes during fluid resuscitation and up to four days postburn., Methods: We conducted an experimental study in patients with a burn injury, as represented by percentage of total body surface area burned (TBSA), of more than 25% in a university eight-bed burns intensive care unit serving about 3.5 million inhabitants. Six patients with a median TBSA percentage of 59% (range 33.5% to 90%) and nine healthy controls were examined by intracutaneous MD, in which recordings of glucose, pyruvate, lactate, glycerol, and urea were performed., Results: Blood glucose concentration peaked on day two at 9.8 mmol/l (6.8 to 14.0) (median and range) and gradually declined on days three and four, whereas skin glucose in MD continued to increase throughout the study period with maximum values on day four, 8.7 mmol/l (4.9 to 11.0). Controls had significantly lower skin glucose values compared with burn patients, 3.1 mmol/l (1.5 to 4.6) (p < 0.001). Lactate from burn patients was significantly higher than controls in both injured and uninjured skin (MD), 4.6 mmol/l (1.3 to 8.9) and 3.8 mmol/l (1.6 to 7.5), respectively (p < 0.01). The skin lactate/pyruvate ratio (MD) was significantly increased in burn patients on all days (p < 0.001). Skin glycerol (MD) was significantly increased at days three and four in burn patients compared with controls (p < 0.01)., Conclusion: Despite a strategy that fulfilled conventional goals for resuscitation, there were increased lactate/pyruvate ratios, indicative of local acidosis. A corresponding finding was not recorded systemically. We conclude that MD is a promising tool for depicting local metabolic processes that are not fully appreciated when examined systemically. Because the local response in glucose, lactate, and pyruvate metabolism seems to differ from that recorded systemically, this technique may offer a new method of monitoring organs.

The purpose of the present study was to identify whether young patients with type 1 diabetes using modern multiple insulin injection therapy (MIT) have signs of microvascular dysfunction and to elucidate possible correlations with various disease parameters. Skin blood flow on the dorsum of the foot was measured with laser Doppler perfusion imaging in 37 patients (age 10-21 years, disease duration 6.0-16 years) and 10 healthy controls. Measurements were performed at rest, after change in posture (the leg was lowered below heart level) and during postocclusive hyperaemia. Following a change in posture blood flow increased instead of decreased in a majority of the study subjects. Patients with acute HbA1c >7.5% (n = 22) had an increase in skin blood flow at rest and a significantly reduced blood flow when the leg was lowered below heart level as compared with patients with HbA1c <7.5% (0.26 V versus 0.17 V, P<0.01 and 0.12 V versus 0.23 V, P<0.05, respectively) and healthy controls. Following occlusion of the macrocirculation for 3 min a small non-significant decrease in the hyperaemic response was seen in the patients. The postocclusive hyperaemic response and the venoarteriolar reflex were not correlated to duration of disease, long-term metabolic control or electrophysiological signs of peripheral nerve dysfunction. It is concluded that signs of microvascular dysfunction related to poor metabolic control are present in young patients with MIT treatment and rather well-controlled diabetes. Low resting blood flow levels are suggested to contribute to the absence of postural vasoconstrictor response.

The distribution of sitting pressure and ability to respond with reactive hyperaemia were studied in a group of paraplegic and tetraplegic patients (n = 8) with spinal cord lesions and healthy controls (n = 10) using a pressure sensitive plate and laser Doppler perfusion imager. The results show that the mean sitting pressure of the patients was 9.9 N/cm2 (left) and 11.7 N/cm2 (right) compared with 3.5 N/cm2 (left) and 3.6 N/cm2 (right) in controls. The differences were significant on both the left (p < 0.01) and right (p < 0.05) sides. The maximum pressure in patients was 42.9 N/cm2 (left) and 48.7 N/cm2 (right), and in controls 12.0 N/cm2 (left) and 12.9 (right) (p < 0.01). Both groups showed a reduction in skin perfusion in the seat area during sitting compared with unloaded resting, and in the controls it was significantly increased (p < 0.001 on both sides) during the reactive hyperaemic phase immediately after sitting. Compared with the preload values, the patients showed a similar but slightly weaker picture significant on the right side (p < 0.05), but not on the left. The hyperaemia was not uniformly distributed, but occurred where the pressure was greater than 2 N/cm2. There was no correlation between the amount of reactive hyperaemia and absolute values of sitting pressures. We conclude that tetraplegic and paraplegic patients have significantly higher sitting pressures than normal controls, and that the hyperaemic response in the buttock region in the upright position after pressure load is slightly weaker in the patients, which could be of importance for the development of decubitus ulcers.