Publications by authors named "Jonathan Brewer"

53 Publications

One-to-one innervation of vocal muscles allows precise control of birdsong.

Curr Biol 2021 May 26. Epub 2021 May 26.

Department of Biology, University of Southern Denmark, Campusvej 55, 5230 Odense, Denmark. Electronic address:

The motor control resolution of any animal behavior is limited to the minimal force step available when activating muscles, which is set by the number and size distribution of motor units (MUs) and muscle-specific force. Birdsong is an excellent model system for understanding acquisition and maintenance of complex fine motor skills, but we know surprisingly little about how the motor pool controlling the syrinx is organized and how MU recruitment drives changes in vocal output. Here we developed an experimental paradigm to measure MU size distribution using spatiotemporal imaging of intracellular calcium concentration in cross-sections of living intact syrinx muscles. We combined these measurements with muscle stress and an in vitro syrinx preparation to determine the control resolution of fundamental frequency (f), a key vocal parameter, in zebra finches. We show that syringeal muscles have extremely small MUs, with 40%-50% innervating ≤3 and 13%-17% innervating a single muscle fiber. Combined with the lowest specific stress (5 mN/mm) known to skeletal vertebrate muscle, small force steps by the major f controlling muscle provide control of 50-mHz to 7.3-Hz steps per MU. We show that the song system has the highest motor control resolution possible in the vertebrate nervous system and suggest this evolved due to strong selection on fine gradation of vocal output. Furthermore, we propose that high-resolution motor control was a key feature contributing to the radiation of songbirds that allowed diversification of song and speciation by vocal space expansion.
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http://dx.doi.org/10.1016/j.cub.2021.05.008DOI Listing
May 2021

In vitro skin model for characterization of sunscreen substantivity upon perspiration.

Int J Cosmet Sci 2021 Jun 9;43(3):359-371. Epub 2021 Jun 9.

Department of Chemistry, Technical University of Denmark, Lyngby, Denmark.

Objective: The resistance of sunscreens to the loss of ultraviolet (UV) protection upon perspiration is important for their practical efficacy. However, this topic is largely overlooked in evaluations of sunscreen substantivity due to the relatively few well-established protocols compared to those for water resistance and mechanical wear.

Methods: In an attempt to achieve a better fundamental understanding of sunscreen behaviour in response to sweat exposure, we have developed a perspiring skin simulator, containing a substrate surface that mimics sweating human skin. Using this perspiring skin simulator, we evaluated sunscreen performance upon perspiration by in vitro sun protection factor (SPF) measurements, optical microscopy, ultraviolet (UV) reflectance imaging and coherent anti-Stokes Raman scattering (CARS) microscopy.

Results And Conclusion: Results indicated that perspiration reduced sunscreen efficiency through two mechanisms, namely sunscreen wash-off (impairing the film thickness) and sunscreen redistribution (impairing the film uniformity). Further, we investigated how the sweat rate affected these mechanisms and how sunscreen application dose influenced UV protection upon perspiration. As expected, higher sweat rates led to a large loss of UV protection, while a larger application dose led to larger amounts of sunscreen being washed-off and redistributed but also provided higher UV protection before and after sweating.
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http://dx.doi.org/10.1111/ics.12703DOI Listing
June 2021

Multiple Na,K-ATPase Subunits Colocalize in the Brush Border of Mouse Choroid Plexus Epithelial Cells.

Int J Mol Sci 2021 Feb 4;22(4). Epub 2021 Feb 4.

Department of Biomedicine, Faculty of Health Science, Aarhus University, 8000 Aarhus, Denmark.

(1) Background: The unusual accumulation of Na,K-ATPase complexes in the brush border membrane of choroid plexus epithelial cells have intrigued researchers for decades. However, the full range of the expressed Na,K-ATPase subunits and their relation to the microvillus cytoskeleton remains unknown. (2) Methods: RT-PCR analysis, co-immunoprecipitation, native PAGE, mass spectrometry, and differential centrifugation were combined with high-resolution immunofluorescence histochemistry, proximity ligase assays, and stimulated emission depletion (STED) microscopy on mouse choroid plexus cells or tissues in order to resolve these issues. (3) Results: The choroid plexus epithelium expresses Na,K-ATPase subunits α1, α2, β1, β2, β3, and phospholemman. The α1, α2, β1, and β2, subunits are all localized to the brush border membrane, where they appear to form a complex. The ATPase complexes may stabilize in the brush border membrane via anchoring to microvillar actin indirectly through ankyrin-3 or directly via other co-precipitated proteins. Aquaporin 1 (AQP1) may form part of the proposed multi-protein complexes in contrast to another membrane protein, the Na-K-2Cl cotransporter 1 (NKCC1). NKCC1 expression seems necessary for full brush border membrane accumulation of the Na,K-ATPase in the choroid plexus. (4) Conclusion: A multitude of Na,K-ATPase subunits form molecular complexes in the choroid plexus brush border, which may bind to the cytoskeleton by various alternative actin binding proteins.
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http://dx.doi.org/10.3390/ijms22041569DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7915972PMC
February 2021

Plasticity of Epididymal Adipose Tissue in Response to Diet-Induced Obesity at Single-Nucleus Resolution.

Cell Metab 2021 02 29;33(2):437-453.e5. Epub 2020 Dec 29.

Center for Functional Genomics and Tissue Plasticity, Department of Biochemistry and Molecular Biology, University of Southern Denmark, Odense M 5230, Denmark. Electronic address:

Adipose tissues display a remarkable ability to adapt to the dietary status. Here, we have applied single-nucleus RNA-seq to map the plasticity of mouse epididymal white adipose tissue at single-nucleus resolution in response to high-fat-diet-induced obesity. The single-nucleus approach allowed us to recover all major cell types and to reveal distinct transcriptional stages along the entire adipogenic trajectory from preadipocyte commitment to mature adipocytes. We demonstrate the existence of different adipocyte subpopulations and show that obesity leads to disappearance of the lipogenic subpopulation and increased abundance of the stressed lipid-scavenging subpopulation. Moreover, obesity is associated with major changes in the abundance and gene expression of other cell populations, including a dramatic increase in lipid-handling genes in macrophages at the expense of macrophage-specific genes. The data provide a powerful resource for future hypothesis-driven investigations of the mechanisms of adipocyte differentiation and adipose tissue plasticity.
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http://dx.doi.org/10.1016/j.cmet.2020.12.004DOI Listing
February 2021

Substituted 9-Diethylaminobenzo[]phenoxazin-5-ones (Nile Red Analogues): Synthesis and Photophysical Properties.

J Org Chem 2021 01 28;86(2):1471-1488. Epub 2020 Dec 28.

Nile Red is a benzo[]phenoxazone dye containing a diethylamino substituent at the 9-position. In recent years, it has become a popular histological stain for cellular membranes and lipid droplets due to its unrivaled fluorescent properties in lipophilic environments. This makes it an attractive lead for chemical decoration to tweak its attributes and optimize it for more specialized microscopy techniques, e.g., fluorescence lifetime imaging or two-photon excited fluorescence microscopy, to which Nile Red has never been optimized. Herein, we present synthesis approaches to a series of monosubstituted Nile Red derivatives (9-diethylbenzo[]phenoxazin-5-ones) starting from 1-naphthols or 1,3-naphthalenediols. The solvatochromic responsiveness of these fluorophores is reported with focus on how the substituents affect the absorption and emission spectra, luminosity, fluorescence lifetimes, and two-photon absorptivity. Several of the analogues emerge as strong candidates for reporting the polarity of their local environment. Specifically, the one- and two-photon excited fluorescence of Nile Red turns out to be very responsive to substitution, and the spectroscopic features can be finely tuned by judiciously introducing substituents of distinct electronic character at specific positions. This new toolkit of 9-diethylbenzo[]phenoxazine-5-ones constitutes a step toward the next generation of optical molecular probes for advancing the understanding of lipid structures and cellular processes.
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http://dx.doi.org/10.1021/acs.joc.0c02346DOI Listing
January 2021

Epidermal Acyl-CoA-binding protein is indispensable for systemic energy homeostasis.

Mol Metab 2021 02 18;44:101144. Epub 2020 Dec 18.

Department of Biochemistry and Molecular Biology, Villum Center for Bioanalytical Sciences, University of Southern Denmark, Campusvej 55, 5230, Odense M, Denmark. Electronic address:

Objectives: The skin is the largest sensory organ of the human body and plays a fundamental role in regulating body temperature. However, adaptive alterations in skin functions and morphology have only vaguely been associated with physiological responses to cold stress or sensation of ambient temperatures. We previously found that loss of acyl-CoA-binding protein (ACBP) in keratinocytes upregulates lipolysis in white adipose tissue and alters hepatic lipid metabolism, suggesting a link between epidermal barrier functions and systemic energy metabolism.

Methods: To assess the physiological responses to loss of ACBP in keratinocytes in detail, we used full-body ACBP and skin-specific ACBP knockout mice to clarify how loss of ACBP affects 1) energy expenditure by indirect calorimetry, 2) response to high-fat feeding and a high oral glucose load, and 3) expression of brown-selective gene programs by quantitative PCR in inguinal WAT (iWAT). To further elucidate the role of the epidermal barrier in systemic energy metabolism, we included mice with defects in skin structural proteins (ma/ma Flg) in these studies.

Results: We show that the ACBP mice and skin-specific ACBP knockout mice exhibited increased energy expenditure, increased food intake, browning of the iWAT, and resistance to diet-induced obesity. The metabolic phenotype, including browning of the iWAT, was reversed by housing the mice at thermoneutrality (30 °C) or pharmacological β-adrenergic blocking. Interestingly, these findings were phenocopied in flaky tail mice (ma/ma Flg). Taken together, we demonstrate that a compromised epidermal barrier induces a β-adrenergic response that increases energy expenditure and browning of the white adipose tissue to maintain a normal body temperature.

Conclusions: Our findings show that the epidermal barrier plays a key role in maintaining systemic metabolic homeostasis. Thus, regulation of epidermal barrier functions warrants further attention to understand the regulation of systemic metabolism in further detail.
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http://dx.doi.org/10.1016/j.molmet.2020.101144DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7797911PMC
February 2021

Drinking and Water Handling in the Medaka Intestine: A Possible Role of Claudin-15 in Paracellular Absorption?

Int J Mol Sci 2020 Mar 8;21(5). Epub 2020 Mar 8.

Department of Biological Sciences, University of Arkansas, SCEN 601, Fayetteville, AR 72701, USA.

When euryhaline fish move between fresh water (FW) and seawater (SW), the intestine undergoes functional changes to handle imbibed SW. In Japanese medaka, the potential transcellular aquaporin-mediated conduits for water are paradoxically downregulated during SW acclimation, suggesting paracellular transport to be of principal importance in hyperosmotic conditions. In mammals, intestinal claudin-15 (CLDN15) forms paracellular channels for small cations and water, which may participate in water transport. Since two paralogs, and , have previously been identified in medaka, we examined the salinity effects on their mRNA expression and immunolocalization in the intestine. In addition, we analyzed the drinking rate and intestinal water handling by adding non-absorbable radiotracers, 51-Cr-EDTA or 99-Tc-DTPA, to the water. The drinking rate was >2-fold higher in SW than FW-acclimated fish, and radiotracer experiments showed anterior accumulation in FW and posterior buildup in SW intestines. Salinity had no effect on expression of , while was approximately 100-fold higher in FW than SW. Despite differences in transcript dynamics, Cldn15a and Cldn15b proteins were both similarly localized in the apical tight junctions of enterocytes, co-localizing with occludin and with no apparent difference in localization and abundance between FW and SW. The stability of the Cldn15 protein suggests a physiological role in water transport in the medaka intestine.
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http://dx.doi.org/10.3390/ijms21051853DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7085193PMC
March 2020

Vibrational Spectroscopic Characterization and Coherent Anti-Stokes Raman Spectroscopy (CARS) Imaging of Artepillin C.

Appl Spectrosc 2020 Jul 30;74(7):751-757. Epub 2020 Apr 30.

Department of Biochemistry and Molecular Biology (BMB), University of Southern Denmark, Odense, Denmark.

In the following work, the vibrational spectroscopic characteristics of artepillin C are reported by means of Fourier transform infrared (FT-IR) and Raman spectroscopies, surface-enhanced Raman scattering (SERS), and coherent anti-Stokes Raman scattering (CARS) microscopy. Artepillin C is an interesting compound due to its pharmacological properties, including antitumor activity. It is found as the major component of Brazilian green propolis, a resinous mixture produced by bees to protect their hives against intruders. Vibrational spectroscopic techniques have shown a strong peak at 1599 cm, assigned to C=C stretching vibrations from the aromatic ring of artepillin C. From these data, direct visualization of artepillin C could be assessed by means of CARS microscopy, showing differences in the film hydration obtained for its neutral and deprotonated states. Raman-based methods show potential to visualize the uptake and action of artepillin C in biological systems, triggering its interaction with biological systems that are needed to understand its mechanism of action.
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http://dx.doi.org/10.1177/0003702820904456DOI Listing
July 2020

Measuring molecular order for lipid membrane phase studies: Linear relationship between Laurdan generalized polarization and deuterium NMR order parameter.

Biochim Biophys Acta Biomembr 2019 12 28;1861(12):183053. Epub 2019 Aug 28.

Department of Physics, Simon Fraser University, Burnaby, BC, Canada; Department of Molecular Biology and Biochemistry, Simon Fraser University, Burnaby, BC, Canada. Electronic address:

Two dimensional phase separation in lipid membranes and cell membranes is of interest to biology because of the idea of membrane rafts - compositionally heterogeneous liquid crystal domains with cellular functions. Few quantitative tools exist for characterizing and differentiating coexisting phases on a molecular scale. Lipid acyl chain order can be measured directly using deuterium nuclear magnetic resonance spectroscopy (H NMR), or inferred using fluorescence microscopy along with the environment-sensitive probe Laurdan. We found a linear relationship between the H NMR order parameter and Laurdan generalized polarization. This observed correlation supports the idea that lipid chain order is tightly associated with the amount and dynamics of water molecules at the glycerol backbone level of the membrane.
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http://dx.doi.org/10.1016/j.bbamem.2019.183053DOI Listing
December 2019

Label free noninvasive spatially resolved NaCl concentration measurements using Coherent Anti-Stokes Raman Scattering microscopy applied to butter.

Food Chem 2019 Nov 23;297:124881. Epub 2019 May 23.

Department of Biochemistry and Molecular Biology, University of Southern Denmark, Odense M, Denmark. Electronic address:

Imaging the microstructure of opaque composite foodstuffs and extracting quantitative chemical information about specific localized components is challenging. Herein, a method has been developed to determine spatially resolved concentrations of aqueous salt and applied to measure salt concentrations of water droplets in butter samples. This was done using Coherent Anti-Stokes Raman Scattering (CARS) microscopy which achieves non-invasive label free imaging based on visualization of specific chemical-bond vibrations. The concentration of salt in the dispersed water droplets in butter was determined based on the relative change in intensity of the CARS-signal at two distinct wavenumbers, which have been shown to be dependent on the inter-molecular coupling of water molecules and salt. The results provide the size and salt concentration distribution of the droplets in the samples. It is further shown that the average salt concentration in the whole sample can correctly be inferred from the concentration measured within the water droplets.
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http://dx.doi.org/10.1016/j.foodchem.2019.05.155DOI Listing
November 2019

Accelerated redevelopment of vocal skills is preceded by lasting reorganization of the song motor circuitry.

Elife 2019 05 17;8. Epub 2019 May 17.

Department of Behavioural Neurobiology, Max Planck Institute for Ornithology, Seewiesen, Germany.

Complex motor skills take considerable time and practice to learn. Without continued practice the level of skill performance quickly degrades, posing a problem for the timely utilization of skilled motor behaviors. Here we quantified the recurring development of vocal motor skills and the accompanying changes in synaptic connectivity in the brain of a songbird, while manipulating skill performance by consecutively administrating and withdrawing testosterone. We demonstrate that a songbird with prior singing experience can significantly accelerate the re-acquisition of vocal performance. We further demonstrate that an increase in vocal performance is accompanied by a pronounced synaptic pruning in the forebrain vocal motor area HVC, a reduction that is not reversed when birds stop singing. These results provide evidence that lasting synaptic changes in the motor circuitry are associated with the savings of motor skills, enabling a rapid recovery of motor performance under environmental time constraints.
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http://dx.doi.org/10.7554/eLife.43194DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6570526PMC
May 2019

Strain-Dependent Structural Changes in Major and Minor Ampullate Spider Silk Revealed by Two-Photon Excitation Polarization.

Biomacromolecules 2019 06 22;20(6):2384-2391. Epub 2019 May 22.

Department of Biochemistry and Molecular Biology , University of Southern Denmark , 5230 Odense , Denmark.

Spider silk's mechanical properties make it an interesting material for many industrial applications. The structure and nanoscopic organization of its proteins are the basis of these qualities. In this study, the emission maxima of the autofluorescence from the protein core of major and minor ampullate silk fibers from the orb-web-weaving spider Nephila madagascariensis are determined and found to be 534 ± 11 and 547 ± 19 nm, respectively. Molecular conformational changes during applied strain are observed in both fiber types using two-photon excitation polarization measurements. Our findings showed that within the fibers the autofluorescent dipoles are separated into two distinct populations, one randomly orientated (amorphous regions) and one with aligned dipoles as found in crystalline structures. The crystalline-amorphous ratio was determined, and it was found that the crystalline dipoles made up around 30 and 20% of the autofluorescent dipoles in major and minor ampullate silk fibers, respectively. Using two-photon polarization measurements, it is possible to directly observe that the major and minor ampullate silk fibers structurally adapt to the applied stress, as well as discern different molecular conformational changes between major and minor ampullates. It was seen that the crystalline-amorphous ratio increased, with up to 9% for major fibers and 6% for minor fibers, as strain was applied, suggesting a conformational adaptation of the fiber, interpreted as noncrystalline 3-helices transforming into crystalline β-sheets.
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http://dx.doi.org/10.1021/acs.biomac.9b00368DOI Listing
June 2019

Assessing Collagen and Elastin Pressure-dependent Microarchitectures in Live, Human Resistance Arteries by Label-free Fluorescence Microscopy.

J Vis Exp 2018 04 9(134). Epub 2018 Apr 9.

Department of Cardiovascular and Renal Research, Institute of Molecular Medicine, University of Southern Denmark; Department of Cardiac, Thoracic and Vascular Surgery, Odense University Hospital.

The pathogenic contribution of resistance artery remodeling is documented in essential hypertension, diabetes and the metabolic syndrome. Investigations and development of microstructurally motivated mathematical models for understanding the mechanical properties of human resistance arteries in health and disease have the potential to aid understanding how disease and medical treatments affect the human microcirculation. To develop these mathematical models, it is essential to decipher the relationship between the mechanical and microarchitectural properties of the microvascular wall. In this work, we describe an ex vivo method for passive mechanical testing and simultaneous label-free three-dimensional imaging of the microarchitecture of elastin and collagen in the arterial wall of isolated human resistance arteries. The imaging protocol can be applied to resistance arteries of any species of interest. Image analyses are described for quantifying i) pressure-induced changes in internal elastic lamina branching angles and adventitial collagen straightness using Fiji and ii) collagen and elastin volume densities determined using Ilastik software. Preferably all mechanical and imaging measurements are performed on live, perfused arteries, however, an alternative approach using standard video-microscopy pressure myography in combination with post-fixation imaging of re-pressurized vessels is discussed. This alternative method provides users with different options for analysis approaches. The inclusion of the mechanical and imaging data in mathematical models of the arterial wall mechanics is discussed, and future development and additions to the protocol are proposed.
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http://dx.doi.org/10.3791/57451DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5933429PMC
April 2018

Dynamic Changes in the Protein Localization in the Nuclear Environment in Pancreatic β-Cell after Brief Glucose Stimulation.

J Proteome Res 2018 04 15;17(4):1664-1676. Epub 2018 Mar 15.

Protein Research Group, Department of Biochemistry and Molecular Biology , University of Southern Denmark , DK-5230 Odense M , Denmark.

Characterization of molecular mechanisms underlying pancreatic β-cell function in relation to glucose-stimulated insulin secretion is incomplete, especially with respect to global response in the nuclear environment. We focus on the characterization of proteins in the nuclear environment of β-cells after brief, high glucose stimulation. We compared purified nuclei derived from β-cells stimulated with 17 mM glucose for 0, 2, and 5 min using quantitative proteomics, a time frame that most likely does not result in translation of new protein in the cell. Among the differentially regulated proteins, we identified 20 components of the nuclear organization processes, including nuclear pore organization, ribonucleoprotein complex, and pre-mRNA transcription. We found alteration of the nuclear pore complex, together with calcium/calmodulin-binding chaperones that facilitate protein and RNA import or export to/from the nucleus to the cytoplasm. Putative insulin mRNA transcription-associated factors were identified among the regulated proteins, and they were cross-validated by Western blotting and confocal immunofluorescence imaging. Collectively, our data suggest that protein translocation between the nucleus and the cytoplasm is an important process, highly involved in the initial molecular mechanism underlying glucose-stimulated insulin secretion in pancreatic β-cells.
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http://dx.doi.org/10.1021/acs.jproteome.7b00930DOI Listing
April 2018

Fundamental constraints in synchronous muscle limit superfast motor control in vertebrates.

Elife 2017 11 22;6. Epub 2017 Nov 22.

Department of Biology, University of Southern Denmark, Odense, Denmark.

Superfast muscles (SFMs) are extremely fast synchronous muscles capable of contraction rates up to 250 Hz, enabling precise motor execution at the millisecond time scale. SFM phenotypes have been discovered in most major vertebrate lineages, but it remains unknown whether all SFMs share excitation-contraction coupling pathway adaptations for speed, and if SFMs arose once, or from independent evolutionary events. Here, we demonstrate that to achieve rapid actomyosin crossbridge kinetics bat and songbird SFM express myosin heavy chain genes that are evolutionarily and ontologically distinct. Furthermore, we show that all known SFMs share multiple functional adaptations that minimize excitation-contraction coupling transduction times. Our results suggest that SFM evolved independently in sound-producing organs in ray-finned fish, birds, and mammals, and that SFM phenotypes operate at a maximum operational speed set by fundamental constraints in synchronous muscle. Consequentially, these constraints set a fundamental limit to the maximum speed of fine motor control.
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http://dx.doi.org/10.7554/eLife.29425DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5699865PMC
November 2017

Developing and testing a comprehensive tool to assess family meetings: Empirical distinctions between high- and low-quality meetings.

J Crit Care 2017 12 28;42:223-230. Epub 2017 Jul 28.

Department of Palliative, Rehabilitation and Integrative Medicine, University of Texas MD Anderson Cancer Center, Houston, TX, USA.

Background: The heterogeneity with regard to findings on family meetings (or conferences) suggests a need to better understand factors that influence family meetings. While earlier studies have explored frequency or timing of family meetings, little is known about how factors (such as what is said during meetings, how it is said, and by whom) influence family meeting quality.

Objectives: (1) To develop an evaluation tool to assess family meetings (Phase 1); (2) to identify factors that influence meeting quality by evaluating 34 family meetings (Phase 2).

Materials And Methods: For Phase 1, methods included developing a framework, cognitive testing, and finalizing the evaluation tool. The tool consisted of Facilitator Characteristics (i.e., gender, experience, and specialty of the person leading the meeting), and 22 items across 6 Meeting Elements (i.e., Introductions, Information Exchanges, Decisions, Closings, Communication Styles, and Emotional Support) and sub-elements. For Phase 2, methods included training evaluators, assessing family meetings, and analyzing data. We used Spearman's rank-order correlations to calculate meeting quality. Qualitative techniques were used to analyze free-text.

Results: No Facilitator Characteristic had a significant correlation with meeting quality. Sub-elements related to communication style and emotional support most strongly correlated with high-quality family meetings, as well as whether "next steps" were outlined (89.66%) and whether "family understanding" was elicited (86.21%). We also found a significant and strong positive association between overall proportion scores and evaluators' ratings (r=0.731, p<0.001).

Conclusions: We filled a gap by developing an evaluation tool to assess family meetings, and we identified how what is said during meetings impacts quality.
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http://dx.doi.org/10.1016/j.jcrc.2017.07.040DOI Listing
December 2017

Imaging and modeling of acute pressure-induced changes of collagen and elastin microarchitectures in pig and human resistance arteries.

Am J Physiol Heart Circ Physiol 2017 Jul 21;313(1):H164-H178. Epub 2017 Apr 21.

Department of Cardiovascular and Renal Research, Institute of Molecular Medicine, University of Southern Denmark, Odense, Denmark.

The impact of disease-related changes in the extracellular matrix (ECM) on the mechanical properties of human resistance arteries largely remains to be established. Resistance arteries from both pig and human parietal pericardium (PRA) display a different ECM microarchitecture compared with frequently used rodent mesenteric arteries. We hypothesized that the biaxial mechanics of PRA mirror pressure-induced changes in the ECM microarchitecture. This was tested using isolated pig PRA as a model system, integrating vital imaging, pressure myography, and mathematical modeling. Collagenase and elastase digestions were applied to evaluate the load-bearing roles of collagen and elastin, respectively. The incremental elastic modulus linearly related to the straightness of adventitial collagen fibers circumferentially and longitudinally (both ≥ 0.99), whereas there was a nonlinear relationship to the internal elastic lamina elastin fiber branching angles. Mathematical modeling suggested a collagen recruitment strain (means ± SE) of 1.1 ± 0.2 circumferentially and 0.20 ± 0.01 longitudinally, corresponding to a pressure of ~40 mmHg, a finding supported by the vital imaging. The integrated method was tested on human PRA to confirm its validity. These showed limited circumferential distensibility and elongation and a collagen recruitment strain of 0.8 ± 0.1 circumferentially and 0.06 ± 0.02 longitudinally, reached at a distending pressure below 20 mmHg. This was confirmed by vital imaging showing negligible microarchitectural changes of elastin and collagen upon pressurization. In conclusion, we show here, for the first time in resistance arteries, a quantitative relationship between pressure-induced changes in the extracellular matrix and the arterial wall mechanics. The strength of the integrated methods invites for future detailed studies of microvascular pathologies. This is the first study to quantitatively relate pressure-induced microstructural changes in resistance arteries to the mechanics of their wall. Principal findings using a pig model system were confirmed in human arteries. The combined methods provide a strong tool for future hypothesis-driven studies of microvascular pathologies.
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http://dx.doi.org/10.1152/ajpheart.00110.2017DOI Listing
July 2017

The diffusion dynamics of PEGylated liposomes in the intact vitreous of the ex vivo porcine eye: A fluorescence correlation spectroscopy and biodistribution study.

Int J Pharm 2017 Apr 4;522(1-2):90-97. Epub 2017 Mar 4.

Department for Micro- and Nanotechnology, Technical University of Denmark, Building 345C, 2800 Kgs. Lyngby, Denmark. Electronic address:

The diffusion dynamics of nanocarriers in the vitreous and the influence of nanocarrier physicochemical properties on these dynamics is an important aspect of the efficacy of intravitreal administered nanomedicines for the treatment of posterior segment eye diseases. Here we use fluorescence correlation spectroscopy (FCS) to determine liposome diffusion coefficients in the intact vitreous (D) of ex vivo porcine eyes using a modified Miyake-Apple technique to minimize the disruption of the vitreous fine structure. We chose to investigate whether the zeta potential of polyethylene glycol functionalized (i.e. PEGylated) liposomes altered liposome in situ diffusion dynamics in the vitreous. Non-PEGylated cationic nanocarriers have previously shown little to no diffusion in the vitreous, whilst neutral and anionic have shown diffusion. The liposomes investigated had diameters below 150nm and zeta potentials ranging from -20 to +12mV. We observed that PEGylated cationic liposomes had significantly lower D values (1.14μms) than PEGylated neutral and anionic liposomes (2.78 and 2.87μms). However, PEGylated cationic liposomes had a similar biodistribution profile across the vitreous to the other systems. These results show that PEGylated cationic liposomes with limited cationic charge can diffuse across the vitreous and indicate that the vitreous as a barrier to nanocarriers (Ø<500nm) is more complicated than simply an electrostatic barrier as previously suggested.
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http://dx.doi.org/10.1016/j.ijpharm.2017.03.003DOI Listing
April 2017

Evidence of proteolipid domain formation in an inner mitochondrial membrane mimicking model.

Biochim Biophys Acta Gen Subj 2017 May 7;1861(5 Pt A):969-976. Epub 2017 Feb 7.

Univ Lyon, Université Claude Bernard Lyon 1, ICBMS - UMR CNRS 5246, MEM2, F-69622 Villeurbanne, France. Electronic address:

Background: Mitochondrial creatine kinase (mtCK) is highly abundant in mitochondria; its quantity is equimolecular to the Adenylic Nucleotide Translocator and represents 1% of the mitochondrial proteins. It is a multitask protein localized in the mitochondria intermembrane space where it binds to the specific cardiolipin (CL) phospholipid. If mtCK was initially thought to be exclusively implicated in energy transfer between mitochondria and cytosol through a mechanism referred to as the phosphocreatine shuttle, several recent studies suggested an additional role in maintaining mitochondria membrane structure.

Methods: To further characterized mtCK binding process we used multiphoton excitation fluorescence microscopy coupled with Giant Unilamellar Vesicles (GUV) and laurdan as fluorescence probe.

Results: We gathered structural and dynamical information on the molecular events occurring during the binding of mtCK to the mitochondria inner membrane. We present the first visualization of mtCK-induced CL segregation on a bilayer model forming micrometer-size proteolipid domains at the surface of the GUV. Those microdomains, which only occurred when CL is included in the lipid mixture, were accompanied by the formation of protein multimolecular assembly, vesicle clamping, and changes in both vesicle curvature and membrane fluidity CONCLUSION: Those results highlighted the importance of the highly abundant mtCK in the lateral organization of the mitochondrial inner membrane.

General Significance: Microdomains were induced in mitochondria-mimicking membranes composed of natural phospholipids without cholesterol and/or sphingolipids differing from the proposed cytoplasmic membrane rafts. Those findings as well as membrane curvature modification were discussed in relation with protein-membrane interaction and protein cluster involvement in membrane morphology.
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http://dx.doi.org/10.1016/j.bbagen.2017.02.001DOI Listing
May 2017

Enzymatic studies on planar supported membranes using a widefield fluorescence LAURDAN Generalized Polarization imaging approach.

Biochim Biophys Acta Biomembr 2017 May 23;1859(5):888-895. Epub 2017 Jan 23.

MEMPHYS - Center for Biomembrane Physics, University of Southern Denmark, Odense, Denmark. Electronic address:

We introduce a custom-built instrument designed to perform fast LAURDAN Generalized Polarization (GP) imaging on planar supported membranes. It is mounted on a widefield fluorescence microscope and allows kinetic analysis of the GP function in the millisecond time scale, largely improving the temporal resolution previously achieved using laser scanning based microscopes. A dedicated protocol to calibrate LAURDAN GP data obtained with charge-coupled device (CCD) cameras as detectors is also presented, enabling reliable assignment of GP values in the field of view. Using this methodology we studied structural and dynamical transformations induced by Sphingomyelinase D (SM-D) on planar supported membranes composed of N-lauroyl sphingomyelin (CSM). GP data show the evolution of an initially compositionally homogeneous symmetric bilayer existing in a single liquid disordered phase, to an intermediate configuration showing coexistence of liquid disordered and solid ordered domains, which are not always in-register across the axial plane of the bilayer. This intermediate state, caused by the transformation of CSM to C-ceramide-1-phosphate in the distal leaflet of the bilayer, evolved to a single solid ordered phase at longer time scales. Additionally, we comparatively studied this system using the membrane fluorophore DiIC The advantages and limitations of both fluorescent dyes are discussed, emphasizing the adequacy of LAURDAN GP imaging to explore this type of membrane phenomena.
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http://dx.doi.org/10.1016/j.bbamem.2017.01.024DOI Listing
May 2017

Biochemical and Bioimaging Evidence of Cholesterol in Acquired Cholesteatoma.

Ann Otol Rhinol Laryngol 2016 Aug 15;125(8):627-33. Epub 2016 Apr 15.

MEMPHYS-Centre for Biomembrane Physics, Department of Biochemistry and Molecular Biology, University of Southern Denmark, Odense M, Denmark

Objectives: To quantify the barrier sterols and image the lipid structures in the matrix of acquired cholesteatoma and compare the distribution with that found in stratum corneum from normal skin, with the goal to resolve their potential influence on cholesteatoma growth.

Methods: High-performance thin-layer chromatography (HPTLC) was used to achieve a quantitative biochemical determination of the sterols. The intercellular lipids were visualized by Coherent Anti-Stokes Raman scattering (CARS) microscopy, which enables label-free imaging of the lipids in intact tissue samples.

Results: The results show that the total lipid content of the cholesteatoma matrix is similar to that of stratum corneum from skin and that the cholesteatoma matrix unquestionably contains cholesterol. The cholesterol content in the cholesteatoma matrix is increased by over 30% (w/w dry weight) compared to the control. The cholesterol sulfate content is below 1% of the total lipids in both the cholesteatoma and the control. Cholesterol ester was reduced by over 30% when compared to the control.

Conclusions: The content of cholesterol in the cholesteatoma matrix is significantly different from that in stratum corneum from skin, and we confirm that the main structure of the cholesteatoma resembles very thick stratum corneum.
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http://dx.doi.org/10.1177/0003489416642784DOI Listing
August 2016

Spatial distribution and activity of Na(+)/K(+)-ATPase in lipid bilayer membranes with phase boundaries.

Biochim Biophys Acta 2016 Jun 16;1858(6):1390-9. Epub 2016 Mar 16.

MEMPHYS - Center for Biomembrane Physics, University of Southern Denmark, DK-5230 Odense M, Denmark; Department of Physics, Chemistry, and Pharmacy, University of Southern Denmark, DK-5230 Odense M, Denmark.

We have reconstituted functional Na(+)/K(+)-ATPase (NKA) into giant unilamellar vesicles (GUVs) of well-defined binary and ternary lipid composition including cholesterol. The activity of the membrane system can be turned on and off by ATP. The hydrolytic activity of NKA is found to depend on membrane phase, and the water relaxation in the membrane on the presence of NKA. By collapsing and fixating the GUVs onto a solid support and using high-resolution atomic-force microscopy (AFM) imaging we determine the protein orientation and spatial distribution at the single-molecule level and find that NKA is preferentially located at lo/ld interfaces in two-phase GUVs and homogeneously distributed in single-phase GUVs. When turned active, the membrane is found to unbind from the support suggesting that the protein function leads to softening of the membrane.
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http://dx.doi.org/10.1016/j.bbamem.2016.03.015DOI Listing
June 2016

Endothelin-1 shifts the mediator of bradykinin-induced relaxation from NO to H2 O2 in resistance arteries from patients with cardiovascular disease.

Br J Pharmacol 2016 05 6;173(10):1653-64. Epub 2016 Apr 6.

Department of Cardiovascular and Renal Research, Institute of Molecular Medicine, University of Southern Denmark, Odense, Denmark.

Background And Purpose: We tested the hypothesis that in resistance arteries from cardiovascular disease (CVD) patients, effects of an endothelium-dependent vasodilator depend on the contractile stimulus.

Experimental Approach: Arteries dissected from parietal pericardium of cardiothoracic surgery patients were studied by myography and imaging techniques. Segments were sub-maximally contracted by K(+) , the TxA2 analogue U46619 or endothelin-1 (ET-1).

Key Results: Relaxing effects of Na-nitroprusside were comparable, but those of bradykinin (BK) were bigger in the presence of ET-1 compared with K(+) or U46619. BK-induced relaxation was (i) abolished by L-NAME in K(+) -contracted arteries, (ii) partly inhibited by L-NAME in the presence of U46619 and (iii) not altered by indomethacin, L-NAME plus inhibitors of small and intermediate conductance calcium-activated K(+) channels, but attenuated by catalase, in ET-1-contracted arteries. This catalase-sensitive relaxation was unaffected by inhibitors of NADPH oxidases or allopurinol. Exogenous H2 O2 caused a larger relaxation of ET-1-induced contractions than those evoked by K(+) or U46619 in the presence of inhibitors of other endothelium-derived relaxing factors. Catalase-sensitive staining of cellular ROS with CellROX Deep Red was significantly increased in the presence of both 1 μM BK and 2 nM ET-1 but not either peptide alone.

Conclusions And Implications: In resistance arteries from patients with CVD, exogenous ET-1 shifts the mediator of relaxing responses to the endothelium-dependent vasodilator BK from NO to H2 O2 and neither NADPH oxidases, xanthine oxidase nor NOS appear to be involved in this effect. This might have consequences for endothelial dysfunction in conditions where intra-arterial levels of ET-1 are enhanced.
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http://dx.doi.org/10.1111/bph.13467DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4842913PMC
May 2016

Superresolution and Fluorescence Dynamics Evidence Reveal That Intact Liposomes Do Not Cross the Human Skin Barrier.

PLoS One 2016 11;11(1):e0146514. Epub 2016 Jan 11.

Advanced bioimaging group/MEMPHYS Center for membrane biophysics, Department of Biochemistry and Molecular Biology, University of Southern Denmark, Odense, Denmark.

In this study we use the combination of super resolution optical microscopy and raster image correlation spectroscopy (RICS) to study the mechanism of action of liposomes as transdermal drug delivery systems in human skin. Two different compositions of liposomes were applied to newly excised human skin, a POPC liposome and a more flexible liposome containing the surfactant sodium cholate. Stimulated emission depletion microscopy (STED) images of intact skin and cryo-sections of skin treated with labeled liposomes were recorded displaying an optical resolution low enough to resolve the 100 nm liposomes in the skin. The images revealed that virtually none of the liposomes remained intact beneath the skin surface. RICS two color cross correlation diffusion measurements of double labeled liposomes confirmed these observations. Our results suggest that the liposomes do not act as carriers that transport their cargo directly through the skin barrier, but mainly burst and fuse with the outer lipid layers of the stratum corneum. It was also found that the flexible liposomes showed a greater delivery of the fluorophore into the stratum corneum, indicating that they functioned as chemical permeability enhancers.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0146514PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4709185PMC
July 2016

Neurostereology protocol for unbiased quantification of neuronal injury and neurodegeneration.

Front Aging Neurosci 2015 31;7:196. Epub 2015 Oct 31.

Department of Neuroscience and Experimental Therapeutics, Texas A&M University Health Science Center, College of Medicine Bryan, TX, USA.

Neuronal injury and neurodegeneration are the hallmark pathologies in a variety of neurological conditions such as epilepsy, stroke, traumatic brain injury, Parkinson's disease and Alzheimer's disease. Quantification of absolute neuron and interneuron counts in various brain regions is essential to understand the impact of neurological insults or neurodegenerative disease progression in animal models. However, conventional qualitative scoring-based protocols are superficial and less reliable for use in studies of neuroprotection evaluations. Here, we describe an optimized stereology protocol for quantification of neuronal injury and neurodegeneration by unbiased counting of neurons and interneurons. Every 20th section in each series of 20 sections was processed for NeuN(+) total neuron and parvalbumin(+) interneuron immunostaining. The sections that contain the hippocampus were then delineated into five reliably predefined subregions. Each region was separately analyzed with a microscope driven by the stereology software. Regional tissue volume was determined by using the Cavalieri estimator, as well as cell density and cell number were determined by using the optical disector and optical fractionator. This protocol yielded an estimate of 1.5 million total neurons and 0.05 million PV(+) interneurons within the rat hippocampus. The protocol has greater predictive power for absolute counts as it is based on 3D features rather than 2D images. The total neuron counts were consistent with literature values from sophisticated systems, which are more expensive than our stereology system. This unbiased stereology protocol allows for sensitive, medium-throughput counting of total neurons in any brain region, and thus provides a quantitative tool for studies of neuronal injury and neurodegeneration in a variety of acute brain injury and chronic neurological models.
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http://dx.doi.org/10.3389/fnagi.2015.00196DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4628120PMC
November 2015

Slow Relaxation of Shape and Orientational Texture in Membrane Gel Domains.

Langmuir 2015 Nov 11;31(46):12699-707. Epub 2015 Nov 11.

Institut Curie, UMR3666 CNRS, U1143 INSERM, 26 rue d'Ulm, 75248 Paris Cedex 05, France.

Gel domains in lipid bilayers are structurally more complex than fluid domains. Growth dynamics can lead to noncircular domains with a heterogeneous orientational texture. Most model membrane studies involving gel domain morphology and lateral organization assume the domains to be static. Here we show that rosette shaped gel domains, with heterogeneous orientational texture and a central topological defect, after early stage growth, undergo slow relaxation. On a time scale of days to weeks domains converge to circular shapes and approach uniform texture. 1,2-Dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) enriched gel domains are grown by cooling 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC):DPPC bilayers into the solid-liquid phase coexistence region and are visualized with fluorescence microscopy. The relaxation of individual domains is quantified through image analysis of time-lapse image series. We find a shape relaxation mechanism which is inconsistent with Ostwald ripening and coalescence as observed in membrane systems with coexisting liquid phases. Moreover, domain texture changes in parallel with the changes in domain shape, and selective melting and growth of particular subdomains cause the texture to become more uniform. We propose a relaxation mechanism based on relocation of lipids from high-energy lattice positions, through evaporation-condensation and edge diffusion, to low-energy positions. The relaxation process is modified significantly by binding Shiga toxin, a bacterial toxin from Shigella dysenteriae, to the membrane surface. Binding alters the equilibrium shape of the gel domains from circular to eroded rosettes with disjointed subdomains. This observation may be explained by edge diffusion while evaporation-condensation is restricted, and it provides further support for the proposed overall relaxation mechanism.
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http://dx.doi.org/10.1021/acs.langmuir.5b03168DOI Listing
November 2015

Preparing giant unilamellar vesicles (GUVs) of complex lipid mixtures on demand: Mixing small unilamellar vesicles of compositionally heterogeneous mixtures.

Biochim Biophys Acta 2015 Dec 28;1848(12):3175-80. Epub 2015 Sep 28.

MEMPHYS - Center for Biomembrane Physics, University of Southern Denmark, DK-5230 Odense M, Denmark; Department of Physics, Chemistry, and Pharmacy, University of Southern Denmark, DK-5230 Odense M, Denmark.

Giant unilamellar vesicles (GUVs) are simple model membrane systems of cell-size, which are instrumental to study the function of more complex biological membranes involving heterogeneities in lipid composition, shape, mechanical properties, and chemical properties. We have devised a method that makes it possible to prepare a uniform sample of ternary GUVs of a prescribed composition and heterogeneity by mixing different populations of small unilamellar vesicles (SUVs). The validity of the protocol has been demonstrated by applying it to ternary lipid mixture of DOPC, DPPC, and cholesterol by mixing small unilamellar vesicles (SUVs) of two different populations and with different lipid compositions. The compositional homogeneity among GUVs resulting from SUV mixing is quantified by measuring the area fraction of the liquid ordered-liquid disordered phases in giant vesicles and is found to be comparable to that in GUVs of the prescribed composition produced from hydration of dried lipids mixed in organic solvent. Our method opens up the possibility to quickly increase and manipulate the complexity of GUV membranes in a controlled manner at physiological buffer and temperature conditions. The new protocol will permit quantitative biophysical studies of a whole new class of well-defined model membrane systems of a complexity that resembles biological membranes with rafts.
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http://dx.doi.org/10.1016/j.bbamem.2015.09.020DOI Listing
December 2015

Enzyme-Free Detection of Mutations in Cancer DNA Using Synthetic Oligonucleotide Probes and Fluorescence Microscopy.

PLoS One 2015 27;10(8):e0136720. Epub 2015 Aug 27.

Nucleic Acid Center, Department of Physics, Chemistry and Pharmacy, University of Southern Denmark, Odense, Denmark.

Background: Rapid reliable diagnostics of DNA mutations are highly desirable in research and clinical assays. Current development in this field goes simultaneously in two directions: 1) high-throughput methods, and 2) portable assays. Non-enzymatic approaches are attractive for both types of methods since they would allow rapid and relatively inexpensive detection of nucleic acids. Modern fluorescence microscopy is having a huge impact on detection of biomolecules at previously unachievable resolution. However, no straightforward methods to detect DNA in a non-enzymatic way using fluorescence microscopy and nucleic acid analogues have been proposed so far.

Methods And Results: Here we report a novel enzyme-free approach to efficiently detect cancer mutations. This assay includes gene-specific target enrichment followed by annealing to oligonucleotides containing locked nucleic acids (LNAs) and finally, detection by fluorescence microscopy. The LNA containing probes display high binding affinity and specificity to DNA containing mutations, which allows for the detection of mutation abundance with an intercalating EvaGreen dye. We used a second probe, which increases the overall number of base pairs in order to produce a higher fluorescence signal by incorporating more dye molecules. Indeed we show here that using EvaGreen dye and LNA probes, genomic DNA containing BRAF V600E mutation could be detected by fluorescence microscopy at low femtomolar concentrations. Notably, this was at least 1000-fold above the potential detection limit.

Conclusion: Overall, the novel assay we describe could become a new approach to rapid, reliable and enzyme-free diagnostics of cancer or other associated DNA targets. Importantly, stoichiometry of wild type and mutant targets is conserved in our assay, which allows for an accurate estimation of mutant abundance when the detection limit requirement is met. Using fluorescence microscopy, this approach presents the opportunity to detect DNA at single-molecule resolution and directly in the biological sample of choice.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0136720PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4552304PMC
May 2016

Elastin organization in pig and cardiovascular disease patients' pericardial resistance arteries.

J Vasc Res 2015 31;52(1):1-11. Epub 2015 Mar 31.

Department of Cardiovascular and Renal Research, Institute of Molecular Medicine, University of Southern Denmark, Odense, Denmark.

Peripheral vascular resistance is increased in essential hypertension. This involves structural changes of resistance arteries and stiffening of the arterial wall, including remodeling of the extracellular matrix. We hypothesized that biopsies of the human parietal pericardium, obtained during coronary artery bypass grafting or cardiac valve replacement surgeries, can serve as a source of resistance arteries for structural research in cardiovascular disease patients. We applied two-photon excitation fluorescence microscopy to study the parietal pericardium and isolated pericardial resistance arteries with a focus on the collagen and elastin components of the extracellular matrix. Initial findings in pig tissue were confirmed in patient biopsies. The microarchitecture of the internal elastic lamina in both the pig and patient pericardial resistance arteries (studied at a transmural pressure of 100 mm Hg) is fiber like, and no prominent external elastic lamina could be observed. This microarchitecture is very different from that in rat mesenteric arteries frequently used for resistance artery research. In conclusion, we add three-dimensional information on the structure of the extracellular matrix in resistance arteries from cardiovascular disease patients and propose further use of patient pericardial resistance arteries for studies of the human microvasculature.
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http://dx.doi.org/10.1159/000376548DOI Listing
July 2015