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Background Burn wound progression is a significant problem as burns initially thought to be superficial can actually become full thickness over time. Cooling is an efficient method to reduce burn wound conversion. However, if the cooling agent is below room temperature, depending on the wound size the patient is at risk of hypothermia. Additionally, tissue perfusion is reduced leading to an aggravation of burn wound progression. We investigated if wound dressings based on non-pre-cooled bacterial nanocellulose (BNC) with a high water content cool a burn just by evaporation and reduce the intradermal damages in the skin. Material and methods In a human ex-vivo model, skin explants underwent contact burns using a 100 °C hot steel block. The burned areas were divided into two groups of which one was cooled with a BNC-based wound dressing. Intradermal temperature probes measured temperature in cooled and uncooled burn sites over 24 h. For histological assessments of the burned areas biopsies were taken at different time points. High mobility group box-1 (HMBG1) staining served as marker for cell vitality and necrosis in the different skin layers. Results Intradermal temperature measurement showed that application of the BNC-based wound dressing reduced temperature significantly in burned skin. This cooling effect resulted in a maximum temperature difference of 6.4 ± 1.9 °C and a significant mean reduction of the area under the curve in the first hour after burn of 62% (p Conclusion Based on our results, BNC-based wound dressings cool a burn. Intradermal temperature as well as thermal damage of the tissue was reduced. The tested BNC-based wound dressing can be used without pre-cooling to cool a burn as well as to reduce the burn BNC-based wound progression through its evaporation cooling effect.

Background Bacterial nanocellulose (BNC) is considered a promising carrier for various substances and novel approaches using BNC in combination with antiseptics are well documented. However, the difference in the molecular weight of these molecules influences their uptake by and release from BNC. Analysing the diffusion of standard molecules with different weight, e.g. dextrans, offers the possibility to investigate the mobility of various molecules. We aimed to test the use of BNC regarding uptake and release of different standard molecules as well as two commercially available antiseptics for possible applications in future wound dressings. Material and methods Diffusion profiles, uptake and release of three FITC-dextran molecules differing in weight as well as octenidine (Octenisept®) and povidone-iodine (Betaisodona®)-based antiseptics were tested with BNC-based wound dressings. Furthermore, the antiseptic efficacy of BNC in combination with antiseptics against Staphylococcus aureus was tested. Results Uptake and release capacity for FITC-dextran molecules showed a molecular weight-dependent mobility from BNC into an agarose gel. The loading capacity of BNC was also inversely proportional to the molecular weight of the antiseptics. The release test for octenidine showed a sustained and prolonged delivery into a solid matrix, whereas povidone-iodine was released faster. Both antiseptic solutions combined with BNC showed a good dose-dependent efficacy against S. aureus. Conclusion Results obtained from the mobility of FITC-dextran molecules in the BNC matrix could open possible applications for the combination of BNC with other molecules for medical applications. Combination of both tested antiseptics with BNC showed to be an efficient approach to control bacterial infections.

Eicosanoids are lipid-mediator molecules with key roles in inflammatory skin diseases, such as psoriasis. Eicosanoids are released close to the source of inflammation, where they elicit local pleiotropic effects and dysregulations. Monitoring inflammatory mediators directly in skin lesions could provide new insights and therapeutic possibilities. Here, we analyzed dermal interstitial fluid samples obtained by dermal open-flow microperfusion in a rat model of skin inflammation. We developed a solid-phase extraction ultra-HPLC/MS/MS method to reliably and precisely analyze small-volume samples and quantified 11 eicosanoids [thromboxane B(2), prostaglandin (PG) E(2), PGD(2), PGF(2α), leukotriene B(4), 15-HETE, 12-HETE, 5-HETE, 12-hydroxyeicosapentaenoic acid, 13-HODE, and 17-hydroxydocosahexaenoic acid]. Our method achieved a median intraday precision of approximately 5% and interday precision of approximately 8%. All calibration curves showed excellent linearity between 0.01 and 50 ng/ml (R(2) > 0.980). In the rat model, eicosanoids were significantly increased in imiquimod-treated inflamed skin sites compared with untreated control sites. Oral treatment with an anti-inflammatory glucocorticoid decreased eicosanoid concentrations. These results show that a combination of tissue-specific sampling with LC/MS analytics is well suited for analyzing small sample volumes from minimally invasive sampling methods such as open-flow microperfusion or microdialysis to study local inflammation and the effect of treatments in skin diseases.

Burn injuries initiate numerous processes such as heat shock response, inflammation and tissue regeneration. Reliable burn models are needed to elucidate the exact sequence of local events to be able to better predict when local inflammation triggers systemic inflammatory processes. In contrast to other ex vivo skin culture approaches, we used fresh abdominal skin explants to introduce contact burn injuries. Histological and ultrastructural analyses confirmed a partial-thickness burn pathology. Gene expression patterns and cytokine production profiles of key mediators of the local inflammation, heat shock response, and tissue regeneration were analyzed for 24 h after burn injury. We found significantly increased expression of factors involved in tissue regeneration and inflammation soon after burn injury. To investigate purely inflammation-mediated reactions we injected lipopolysaccharide into the dermis. In comparison to burn injury, lipopolysaccharide injection initiated an inflammatory response while expression patterns of heat shock and tissue regeneration genes were unaffected for the duration of the experiment. This novel ex vivo human skin model is suitable to study the local, early responses to skin injuries such as burns while maintaining an intact overall tissue structure and it gives valuable insights into local mechanisms at the very beginning of the wound healing process after burn injuries.

To investigate the difference in clinical efficacy in AD patients between two topical PDE4 inhibitors using dermal open flow microperfusion and cAMP as a pharmacodynamic read-out in fresh human skin explants. Clinical formulations were applied to intact or barrier disrupted human skin explants and both skin biopsy samples and dermal interstitial fluid was sampled for measuring drug concentration. Furthermore, cAMP levels were determined in the skin biopsies as a measure of target engagement. Elevated cAMP levels were observed with LEO 29102 while no evidence of target engagement was obtained with LEO 39652. In barrier impaired skin the dISF concentration of LEO 29102 was 2100 nM while only 33 nM for LEO 39652. For both compounds the concentrations measured in skin punch biopsies were 7–33-fold higher than the dISF concentrations. Low unbound drug concentration in dISF in combination with minimal target engagement of LEO 39652 in barrier impaired human skin explants supports that lack of clinical efficacy of LEO 39652 in AD patients is likely due to insufficient drug availability at the target. We conclude that dOFM together with a pharmacodynamic target engagement biomarker are strong techniques for establishing skin PK/PD relations and that skin biopsies should be used with caution.

Purpose Dermal open flow microperfusion (dOFM) has previously demonstrated its utility to assess the bioequivalence (BE) of topical drug products in a clinical study. We aimed to characterize the sources of variability in the dermal pharmacokinetic data from that study. Methods Exploratory statistical analyses were performed with multivariate data from a clinical dOFM-study in 20 healthy adults evaluating the BE, or lack thereof, of Austrian test (T) and U.S. reference (R) acyclovir cream, 5% products. Results The overall variability of logAUC values (CV: 39% for R and 45% for T) was dominated by inter-subject variability (R: 82%, T: 91%) which correlated best with the subject’s skin conductance. Intra-subject variability was 18% (R) and 9% (T) of the overall variability; skin treatment sites or methodological factors did not significantly contribute to that variability. Conclusions Inter-subject variability was the major component of overall variability for acyclovir, and treatment site location did not significantly influence intra-subject variability. These results support a dOFM BE study design with T and R products assessed simultaneously on the same subject, where T and R treatment sites do not necessarily need to be next to each other. Localized variation in skin microstructure may be primarily responsible for intra-subject variability.

Defects in autophagy and the resulting deposition of protein aggregates have been implicated in aging and neurodegenerative diseases. While gene targeting in the mouse has facilitated the characterization of these processes in different types of neurons, potential roles of autophagy and accumulation of protein substrates in neuroepithelial cells have remained elusive. Here we report that Atg7f/f Tyr-Cre mice, in which autophagy-related 7 (Atg7) is conditionally deleted under the control of the tyrosinase promoter, are a model for accumulations of the autophagy adapter and substrate sequestosome-1/p62 in both neuronal and neuroepithelial cells. In the brain of Atg7f/f Tyr-Cre but not of fully autophagy competent control mice, p62 aggregates were present in sporadic neurons in the cortex and other brain regions as well in epithelial cells of the choroid plexus and the ependyma. Western blot analysis confirmed a dramatic increase of p62 abundance and formation of high-molecular weight species of p62 in the brain of Atg7f/f Tyr-Cre mice relative to Atg7f/f controls. Immuno-electron microscopy showed that p62 formed filamentous aggregates in neurons and ependymal cells. p62 aggregates were also highly abundant in the ciliary body in the eye. Atg7f/f Tyr-Cre mice reached an age of more than 2 years although neurological defects manifesting in abnormal hindlimb clasping reflexes were evident in old mice. These results show that p62 filaments form in response to impaired autophagy in vivo and suggest that Atg7f/f Tyr-Cre mice are a model useful to study the long-term effects of autophagy deficiency on the homeostasis of different neuroectoderm-derived cells. Electronic supplementary material The online version of this article (10.1007/s12035-018-0996-x) contains supplementary material, which is available to authorized users.

(2)H2O as nonradioactive, stable marker substance is commonly used in preclinical and clinical studies and the precise determination of (2)H2O concentration in biological samples is crucial. However, aside from isotope ratio mass spectrometry (IRMS), only a very limited number of methods to accurately measure the (2)H2O concentration in biological samples are routinely established until now. In this study, we present a straightforward method to accurately measure (2)H-enrichment of rat brain interstitial fluid (ISF) and rat plasma to determine the relative recovery of a cerebral open flow microperfusion (cOFM) probe, using headspace-gas-chromatography - quadrupole-mass-spectrometry. This method is based on basic-catalyzed hydrogen/deuterium exchange in acetone and detects the (2)H-labelled acetone directly by the headspace GC-MS. Small sample volumes and limited number of preparation steps make this method highly competitive. It has been fully validated. (2)H enriched to 8800 ppm in plasma showed an accuracy of 98.9% and %Relative Standard Deviation (RSD) of 3.1 with n = 18 over three days and with two operators. Similar performance was obtained for cerebral ISF enriched to 1100 ppm (accuracy: 96.5%, %RSD: 3.1). With this highly reproducible method we demonstrated the successful employment of (2)H2O as performance marker for a cOFM probe.

Cerebral open flow microperfusion (cOFM) is a new in-vivo technique for continuous sampling of the interstitial fluid in brain tissue. cOFM can be used to monitor substance transport across the blood-brain barrier (pharmacokinetics) and to investigate metabolic changes in brain tissue after drug application (pharmacodynamics). The possibility of long-term implantation into the brain makes cOFM an outstanding tool in the development of brain relevant pharmaceutics.

Background With the increase of antimicrobial resistance in recent decades, other methods of preventing and fighting infections must be considered. Burn patients, whose wound areas are often extensive, are especially prone to wound infections. The loading of bacterial nanocellulose (BNC) with antiseptics has already been successfully performed but unfortunately, the described procedure is time-consuming and thus not applicable in a clinical emergency setting. Therefore, a clinically feasible approach was established. Material and methods Sheets of BNC-based wound dressings were placed into antiseptic solutions containing PHMB (Prontosan® and LAVANID® 2) and were left to soak for up to two hours. At different time points, samples were analysed for their concentration of PHMB and antiseptic efficacy. Results Within 30 min, clinically relevant concentrations of PHMB were achieved in the BNC-based wound dressing. The 30-min PHMB uptake for Prontosan® and LAVANID® 2 resulted in concentrations of 0.05% and 0.019%, respectively. Samples from the PHMB loaded dressing showed a dose dependent antiseptic efficacy for Staphylococcus aureus. Conclusion This experiment showed that the loading of BNC-based wound dressings with PHMB-containing antiseptics was achieved by a simple and quick procedure. According to studies a PHMB concentration of 0.001% can already inhibits all bacterial growth, indicating that the concentrations of PHMB in the BNC-based wound dressings after 30 min are higher than the minimal inhibitory concentration and the antiseptic efficacy after 120 min loading analysed by an standardized bacterial disk diffusion assay was shown to be comparable to the clinically used Suprasorb® X + PHMB wound dressing.

Background Assessment of drug concentration in the brain interstitial fluid (ISF) is crucial for development of brain active drugs, which are mainly small, lipophilic substances able to cross the blood-brain barrier (BBB). We aimed to compare the applicability of cerebral Open Flow Microperfusion (cOFM) and Microdialysis (MD) to sample the lipophilic substance amitriptyline (AMI), its metabolites Hydroxyamitriptyline (HYA), Nortriptyline (NOR), Amitriptyline-N-Oxide (ANO), deuterated water (D2O) and the hydrophilic substance sodium fluorescein (Naf) in brain ISF. New method cOFM has been refined to yield increased spatial resolution and performance. Comparison of cOFM and MD and results Performance of cOFM and MD was assessed by in vivo AUC ratios of probe samples (AUCCOFM/AUCMD) and the in vivo relative recovery of D2O (RRvv,D2O). Adsorption of AMI and Naf to MD and cOFM was assessed by the in vitro relative recovery (RRvt) prior to the in vivo experiments. The in vivo AUC ratio of AMI and RRvv,D2O was about two times higher for cOFM than for MD (AUCOFM/AUCMD = 2.0, RRvv,D2O(cOFM)/RRvv,D2O(MD) = 2.1). cOFM detected all investigated AMI metabolites except NOR. MD did not detect HYA, NOR, ANO and Naf. In vitro adsorption of AMI and Naf to the MD membrane was strong (RRvt,AMI = 4.4%, RRvt,Naf = 1.5%) but unspecific adsorption to cOFM was negligibly small (RRvt,AMI = 98% and RRvt,Naf = 98%). Conclusions cOFM showed better performance when sampling AMI and its metabolites, Naf and D2O, and had an about two times higher RRvv,D2O than MD. MD did not detect HYA, NOR, ANO and Naf, most likely due to membrane adsorption.

Objective The inability of leptin to suppress food intake in diet-induced obesity, sometimes referred to as leptin resistance, is associated with several distinct pathological hallmarks. One prevailing theory is that impaired transport of leptin across the blood–brain barrier (BBB) represents a molecular mechanism that triggers this phenomenon. Recent evidence, however, has challenged this notion, suggesting that leptin BBB transport is acquired during leptin resistance. Methods To resolve this debate, we utilized a novel cerebral Open Flow Microperfusion (cOFM) method to examine leptin BBB transport in male C57BL/6J mice, fed a chow diet or high fat diet (HFD) for 20 days. Results Basal plasma leptin levels were 3.8-fold higher in HFD-fed mice (p, Highlights • Cerebral Open Flow Microperfusion can be utilized to assess hypothalamic leptin levels. • Twenty days of high-fat diet feeding causes functional resistance to exogenous leptin. • Transport of leptin across the BBB is not impaired by HFD-induced leptin resistance.

Extracellular vesicles (EVs) and their miRNA cargo are intercellular communicators transmitting their pleiotropic messages between different cell types, tissues, and body fluids. Recently, they have been reported to contribute to skin homeostasis and were identified as members of the senescence-associated secretory phenotype of human dermal fibroblasts. However, the role of EV-miRNAs in paracrine signaling during skin aging is yet unclear. Here we provide evidence for the existence of small EVs in the human skin and dermal interstitial fluid using dermal open flow microperfusion and show that EVs and miRNAs are transferred from dermal fibroblasts to epidermal keratinocytes in 2D cell culture and in human skin equivalents. We further show that the transient presence of senescent fibroblast derived small EVs accelerates scratch closure of epidermal keratinocytes, whereas long-term incubation impairs keratinocyte differentiation in vitro. Finally, we identify vesicular miR-23a-3p, highly secreted by senescent fibroblasts, as one contributor of the EV-mediated effect on keratinocytes in in vitro wound healing assays. To summarize, our findings support the current view that EVs and their miRNA cargo are members of the senescence-associated secretory phenotype and, thus, regulators of human skin homeostasis during aging.

Drugs for neurological diseases have to cross the blood-brain barrier (BBB) to induce their therapeutic effect. In vivo drug quantification in the brain is challenging, because invasive methods damage the BBB and measurement results may be confounded by drug leakage from the blood into the brain through the disrupted BBB. Cerebral open flow microperfusion (cOFM) is an in vivo sampling technique that allows BBB healing and re-establishment after probe implantation and before sampling is performed. It therefore provides the opportunity to sample compounds in cerebral interstitial fluid with an intact BBB. This article comprehensively describes the experimental setup and procedures, perfusate requirements, critical parameters, common problems that may occur, and their causes and solutions. Typical results from a cOFM sampling experiment are presented and discussed. This protocol provides a tool for performing pharmacokinetic and pharmacodynamic studies in mouse or rat brain with an intact BBB. © 2019 by John Wiley & Sons, Inc.

OBJECTIVE We investigated the impact of two different injection strategies on the pharmacokinetics and pharmacodynamics of insulin aspart in vivo in an open-label, two-period crossover study and verified changes in the surface-to-volume ratio ex vivo. RESEARCH DESIGN AND METHODS Before the clinical trial, insulin aspart was injected ex vivo into explanted human abdominal skin flaps. The surface-to-volume ratio of the subcutaneous insulin depot was assessed by microfocus computed tomography that compared 1 bolus of 18 IU with 9 dispersed boluses of 2 IU. These two injection strategies were then tested in vivo, in 12 C-peptide–negative type 1 diabetic patients in a euglycemic glucose clamp (glucose target 5.5 ± 1.1 mmol/L) for 8 h after the first insulin administration. RESULTS The ex vivo experiment showed a 1.8-fold higher mean surface-to-volume ratio for the dispersed injection strategy. The maximum glucose infusion rates (GIR) were similar for the two strategies (10 ± 4 vs. 9 ± 4; P = 0.5); however, times to reach maximum GIR and 50% and 10% of the maximum GIR were significantly reduced by using the 9 × 2 IU strategy (68 ± 33 vs. 127 ± 93 min; P = 0.01; 38 ± 9 vs. 49 ± 16 min; P < 0.01; 23 ± 6 vs. 30 ± 10 min; P < 0.05). For 9 × 2 IU, the area under the GIR curve was greater during the first 60 min (219 ± 89 vs. 137 ± 75; P < 0.01) and halved until maximum GIR (242 ± 183 vs. 501 ± 396; P < 0.01); however, it was similar across the whole study period (1,361 ± 469 vs. 1,565 ± 527; P = 0.08). CONCLUSIONS A dispersed insulin injection strategy enhanced the effect of a fast-acting insulin analog. The increased surface-to-volume ratio of the subcutaneous insulin depot can facilitate insulin absorption into the vascular system.

Background The availability of generic topical dermatological drug products is constrained by the limited methods established to assess topical bioequivalence (BE). A novel cutaneous pharmacokinetic approach, dermal open-flow microperfusion (dOFM), can continuously assess the rate and extent to which a topical drug becomes available in the dermis, to compare in vivo dermal bioavailability (BA) and support BE evaluations for topical products. Objective To evaluate whether dOFM is an accurate, sensitive, and reproducible in vivo method to characterize the intradermal BA of acyclovir from 5 % acyclovir creams, comparing a reference (R) product either to itself or to a different test (T) product. Methods In a single-center clinical study, R or T products were applied to six randomized treatment sites on the skin of 20 healthy human subjects. Two dOFM probes were inserted in each treatment site to monitor the intradermal acyclovir concentration for 36 h. Comparative BA (of R vs. R and T vs. R) was evaluated based on conventional BE criteria for pharmacokinetic endpoints (area under the curve and maximum plasma concentration) where the 90 % confidence interval of the geometric mean ratio between the T and R falls within 0.80–1.25. Results The positive control products (R vs. R) were accurately and reproducibly confirmed to be bioequivalent, while the negative control products (T vs. R) were sensitively discriminated not to be bioequivalent. Conclusions dOFM accurately, sensitively, and reproducibly characterized the dermal BA in a manner that can support BE evaluations for topical acyclovir 5 % creams in a study with n = 40 (20 subjects in this study).

The availability of generic topical dermatological drug products is constrained by the limited methods established to assess topical bioequivalence (BE). A novel cutaneous pharmacokinetic approach, dermal open-flow microperfusion (dOFM), can continuously assess the rate and extent to which a topical drug becomes available in the dermis, to compare in vivo dermal bioavailability (BA) and support BE evaluations for topical products. To evaluate whether dOFM is an accurate, sensitive, and reproducible in vivo method to characterize the intradermal BA of acyclovir from 5 % acyclovir creams, comparing a reference (R) product either to itself or to a different test (T) product. In a single-center clinical study, R or T products were applied to six randomized treatment sites on the skin of 20 healthy human subjects. Two dOFM probes were inserted in each treatment site to monitor the intradermal acyclovir concentration for 36 h. Comparative BA (of R vs. R and T vs. R) was evaluated based on conventional BE criteria for pharmacokinetic endpoints (area under the curve and maximum plasma concentration) where the 90 % confidence interval of the geometric mean ratio between the T and R falls within 0.80–1.25. The positive control products (R vs. R) were accurately and reproducibly confirmed to be bioequivalent, while the negative control products (T vs. R) were sensitively discriminated not to be bioequivalent. dOFM accurately, sensitively, and reproducibly characterized the dermal BA in a manner that can support BE evaluations for topical acyclovir 5 % creams in a study with n = 40 (20 subjects in this study).