Ketamine reduces electrophysiological network activity in cortical neuron cultures already at sub-micromolar concentrations - Impact on TrkB-ERK1/2 signaling.

The dissociative anesthetic ketamine regulates cortical activity in a dose-dependent manner. Subanesthetic-dose ketamine has paradoxical excitatory effects which is proposed to facilitate brain-derived neurotrophic factor (BDNF) (a ligand of tropomyosin receptor kinase B, TrkB) signaling, and activation of extracellular signal-regulated kinase 1/2 (ERK1/2). Previous data suggests that ketamine, at sub-micromolar concentrations, induces glutamatergic activity, BDNF release, and activation of ERK1/2 also on primary cortical neurons. We combined western blot analysis with multiwell-microelectrode array (mw-MEA) measurements to examine ketamine's concentration-dependent effects on network-level electrophysiological responses and TrkB-ERK1/2 phosphorylation in rat cortical cultures at 14 days in vitro. Ketamine did not cause an increase in neuronal network activity at sub-micromolar concentrations, but instead a decrease in spiking that was evident already at 500 nM concentration. TrkB phosphorylation was unaffected by the low concentrations, although BDNF elicited prominent phosphorylation response. High concentration of ketamine (10 µM) strongly reduced spiking, bursting and burst duration, which was accompanied with decreased phosphorylation of ERK1/2 but not TrkB. Notably, robust increases in spiking and bursting activity could be produced with carbachol, while it did not affect phosphorylation of TrkB or ERK1/2. Diazepam abolished neuronal activity, which was accompanied by reduced ERK1/2 phosphorylation without change on TrkB. In conclusion, sub-micromolar ketamine concentrations did not cause an increase in neuronal network activity or TrkB-ERK1/2 phosphorylation in cortical neuron cultures that readily respond to exogenously applied BDNF. Instead, pharmacological inhibition of network activity can be readily observed with high concentration of ketamine and it is associated with reduced ERK1/2 phosphorylation.

Sensitivity Analysis of Circular and Helmet Coil Arrays in Magnetic Induction Tomography for Stroke Detection.

Magnetic induction tomography (MIT) is harmless and contactless technique for measuring the conductivity of the biological tissue. MIT could be used for initial diagnosis and continuous monitoring of stroke. Different kinds of coil arrays have been proposed for MIT systems. Previous research results using a circular 16-channel MIT model reported difficulties with detection and measurement of small bioelectric signals. For stroke imaging, a system with a higher sensitivity is required. We aim to improve the sensitivity by increasing the number of coils and placing them closer to the head. In this paper, a helmet type coil array with 31 coils is introduced. For simplicity, the head is modelled as a sphere with white matter as a material. The stroke is simulated as a single inclusion with blood and assigned different sizes and positions. Sensitivity distribution and target response of the stroke were evaluated for the helmet model and compared with the circular MIT system. The simulations and analysis were performed at 10 MHz frequency with different coil pairs. Results from comparison of the two MIT models show that the Helmet coil array provides better spatial sensitivity, which has been estimated to be more than 20 times higher than the circular model. Further, when all coils are taken in account, the recorded sensitivity improvement was in the range of 13-90-fold.

Retrieval of the conductivity spectrum of tissuesin vitrowith novel multimodal tomography.

OBJECTIVE: Imaging of tissue engineered three-dimensional (3D) specimens is challenging due to their thickness. We propose a novel multimodal imaging technique to obtain multi-physical 3D images and the electrical conductivity spectrum of tissue engineered specimensin vitro. APPROACH: We combine simultaneous recording of rotational multifrequency electrical impedance tomography (R-mfEIT) with optical projection tomography (OPT). Structural details of the specimen provided by OPT are used here as geometrical priors for R-mfEIT. MAIN RESULTS: This data fusion enables accurate retrieval of the conductivity spectrum of the specimen. We demonstrate experimentally the feasibility of the proposed technique using a potato phantom, adipose and liver tissues, and stem cells in biomaterial spheroids. The results indicate that the proposed technique can distinguish between viable and dead tissues and detect the presence of stem cells. SIGNIFICANCE: This technique is expected to become a valuable tool for monitoring tissue engineered specimens' growth and viabilityin vitro.

Cell adhesion and culture medium dependent changes in the high frequency mechanical vibration induced proliferation, osteogenesis, and intracellular organization of human adipose stem cells.

High frequency (HF) mechanical vibration appears beneficial for in vitro osteogenesis of mesenchymal stem cells (MSCs). However, the current mechanobiological understanding of the method remains insufficient. We designed high-throughput stimulators to apply horizontal or vertical high magnitude HF (HMHF; 2.5 Gpeak, 100 Hz) vibration on human adipose stem cells (hASCs). We analyzed proliferation, alkaline phosphatase (ALP) activity, mineralization, and effects on the actin cytoskeleton and nuclei using immunocytochemical stainings. Proliferation was studied on a standard tissue culture plastic (sTCP) surface and on an adhesion supporting tissue culture plastic (asTCP) surface in basal (BM) and osteogenic (OM) culture medium conditions. We discovered that the improved cell adhesion was a prerequisite for vibration induced changes in the proliferation of hASCs. Similarly, the adhesion supporting surface enabled us to observe vibration initiated ALP activity and mineralization changes in OM condition. The horizontal vibration increased ALP activity, while vertical stimulation reduced ALP activity. However, mineralization was not enhanced by the HMHF vibration. We performed image-based analysis of actin and nuclei to obtain novel data of the intracellular-level responses to HF vibration in BM and OM conditions. Our quantitative results suggest that actin organizations were culture medium and stimulation direction dependent. Both stimulation directions decreased OM induced changes in nuclear size and elongation. Consequently, our findings of the nuclear deformations provide supportive evidence for the involvement of the nuclei in the mechanocoupling of HF vibration. Taken together, the results of this study enhanced the knowledge of the intracellular mechanisms of HF vibration induced osteogenesis of MSCs.

Characterisation and in vitro and in vivo evaluation of supercritical-CO2-foamed ß-TCP/PLCL composites for bone applications.

Most synthetic bone grafts are either hard and brittle ceramics or paste-like materials that differ in applicability from the gold standard autologous bone graft, which restricts their widespread use. Therefore, the aim of the study was to develop an elastic, highly porous and biodegradable ß-tricalciumphosphate/poly(L-lactide-co-ε-caprolactone) (ß-TCP/PLCL) composite for bone applications using supercritical CO2 foaming. Ability to support osteogenic differentiation was tested in human adipose stem cell (hASC) culture for 21 d. Biocompatibility was evaluated for 24 weeks in a rabbit femur-defect model. Foamed composites had a high ceramic content (50 wt%) and porosity (65-67 %). After 50 % compression, in an aqueous environment at 37 °C, tested samples returned to 95 % of their original height. Hydrolytic degradation of ß-TCP/PLCL composite, during the 24-week follow-up, was very similar to that of porous PLCL scaffold both in vitro and in vivo. Osteogenic differentiation of hASCs was demonstrated by alkaline phosphatase activity analysis, alizarin red staining, soluble collagen analysis, immunocytochemical staining and qRT-PCR. In vitro, hASCs formed a pronounced mineralised collagen matrix. A rabbit femur defect model confirmed biocompatibility of the composite. According to histological Masson-Goldner's trichrome staining and micro-computed tomography, ß-TCP/PLCL composite did not elicit infection, formation of fibrous capsule or cysts. Finally, native bone tissue at 4 weeks was already able to grow on and in the ß-TCP/PLCL composite. The elastic and highly porous ß-TCP/PLCL composite is a promising bone substitute because it is osteoconductive and easy-to-use and mould intraoperatively.

Augmenting Soft Tissue Contrast Using Edge-Enhancing Phase-Imaging Techniques in X-Ray Microtomography.

Conventional X-ray imaging is based on the attenuation of X-rays and the technique provides sufficient contrast when the difference between attenuation coefficients of neighboring structures is sufficient. A promising imaging possibility on a µCT is the use of phase information of an X-ray beam to generate an image of the sample. This is known as phase-contrast imaging. Propagation-based phase imaging sets the least amount of requirements on the imaging setup - lateral coherence for the X-ray source and a movable detector and source. The Zeiss Xradia MicroXCT-400 at our laboratory provides this possibility. Therefore, the phase-contrast imaging protocol, which provides an edge-enhancement effect, on the µCT device was optimized using thin polylactic acid fibers in order to enhance the visibility of low density samples. The optimization consisted of source and detector distance variation measurements. To demonstrate the contrast enhancement results, the optimization was applied to two types of collagen samples embedded in air, ethanol, and water.The results showed enhanced contrast for the edge-enhanced phase-contrast images compared to absorption images. Most importantly, the results indicated that the source does not need to placed at the negative limit to obtain useful phase information. Additionally, the visibility increases with increasing sample-to-detector distance. Finally, significantly enhanced contrast was obtained for the collagen sample embedded in water using phase-imaging techniques. The technique is limited due to the focal spot size and voltage of the X-ray source. The phase-imaging technique has the possibility to enhance contrast of low density samples and to reveal structures that cannot be seen using other imaging techniques.

Polypyrrole-coated electrodes show thickness-dependent stability in different conditions during 42-day follow-up in vitro.

Electroconductive polypyrrole/dodecylbenzenesulphonate (PPy/DBS) has been proposed as novel electrode coating for biomedical applications. However, as yet, little is known about its long-term stability in moist conditions. This study compares the stability of PPy/DBS-coated platinum electrodes that are either dry-stored, incubated, or both incubated and electrically stimulated. The electrical and material properties of three different coating thicknesses were monitored for 42 days. Initially, the PPy/DBS-coating decreased the low frequency impedance of the platinum electrodes by 52% to 79%. The dry-stored electrodes remained stable during the follow-up, whereas the properties of all the incubated electrodes were altered in three stages with thickness-dependent duration: stabilization, stable, and degradation. The coated electrodes would be applicable for short-term, low-frequency in vitro measurements of up to 14 days without electrical stimulation, and up to 7 days with stimulation. The coating thickness is bound to other coating properties, and should therefore be selected according to the specific target application. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 106B: 2202-2213, 2018.

Simultaneous Measurement of Contraction and Calcium Transients in Stem Cell Derived Cardiomyocytes.

Induced pluripotent stem cell derived cardiomyocytes (iPSC-CM) provide a powerful platform for disease modeling and drug development in vitro. Traditionally, electrophysiological methods or fluorescent dyes (e.g. calcium) have been used in their functional characterization. Recently, video microscopy has enabled non-invasive analysis of CM contractile motion. Simultaneous assessments of motion and calcium transients have not been generally conducted, as motion detection methods are affected by changing pixel intensities in calcium imaging. Here, we present for the first time a protocol for simultaneous video-based measurement of contraction and calcium with fluorescent dye Fluo-4 videos without corrections, providing data on both ionic and mechanic activity. The method and its accuracy are assessed by measuring the effect of fluorescence and background light on transient widths and contraction velocity amplitudes. We demonstrate the method by showing the contraction-calcium relation and measuring the transient time intervals in catecholaminergic polymorphic ventricular tachycardia patient specific iPSC-CMs and healthy controls. Our validation shows that the simultaneous method provides comparable data to combined individual measurements, providing a new tool for measuring CM biomechanics and calcium simultaneously. Our results with calcium sensitive dyes suggest the method could be expanded to use with other fluorescent reporters as well.

Crystallization and sintering of borosilicate bioactive glasses for application in tissue engineering.

Typical silicate bioactive glasses are known to crystallize readily during the processing of porous scaffolds. While such crystallization does not fully suppress the bioactivity, the presence of significantly large amounts of crystals leads to a decrease in the rate of reaction of the glass and an uncontrolled release of ions. Furthermore, due to the non-congruent dissolution of silicate glasses, these materials have been shown to remain within the surgical site even 14 years post-operation. Therefore, bioactive materials that can dissolve more effectively and have higher conversion rates are required. Within this work, boron was introduced, in the FDA approved S53P4 glass, at the expense of SiO2. The crystallization and sintering-ability of the newly developed glasses were investigated by differential thermal analysis. All the glasses were found to crystallize primarily from the surface, and the crystal phase precipitation was dependent on the quantity of B2O3 incorporated. The rate of crystallization was found to be lower for the glasses when 25, 50 and 75% of SiO2 was replaced with B2O3. These glasses were further sintered into porous scaffolds using simple heat sintering. The impact of glass particle size and heat treatment temperature on the scaffold porosity and average pore size was investigated. Scaffolds with porosity ranging from 10 to 60% and compressive strength ranging from 1 to 35 MPa were produced. The scaffolds remained amorphous during processing and their ability to rapidly precipitate hydroxycarbonate apatite was maintained. This is of particular interest in the field of tissue engineering as scaffold degradation and reaction is generally faster and offers higher controllability as opposed to the current partially/fully crystallized scaffolds obtained from the FDA approved bioactive glasses.

Human induced pluripotent stem cell-derived versus adult cardiomyocytes: an in silico electrophysiological study on effects of ionic current block.

BACKGROUND AND PURPOSE: Two new technologies are likely to revolutionize cardiac safety and drug development: in vitro experiments on human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) and in silico human adult ventricular cardiomyocyte (hAdultV-CM) models. Their combination was recently proposed as a potential replacement for the present hERG-based QT study for pharmacological safety assessments. Here, we systematically compared in silico the effects of selective ionic current block on hiPSC-CM and hAdultV-CM action potentials (APs), to identify similarities/differences and to illustrate the potential of computational models as supportive tools for evaluating new in vitro technologies. EXPERIMENTAL APPROACH: In silico AP models of ventricular-like and atrial-like hiPSC-CMs and hAdultV-CM were used to simulate the main effects of four degrees of block of the main cardiac transmembrane currents. KEY RESULTS: Qualitatively, hiPSC-CM and hAdultV-CM APs showed similar responses to current block, consistent with results from experiments. However, quantitatively, hiPSC-CMs were more sensitive to block of (i) L-type Ca(2+) currents due to the overexpression of the Na(+) /Ca(2+) exchanger (leading to shorter APs) and (ii) the inward rectifier K(+) current due to reduced repolarization reserve (inducing diastolic potential depolarization and repolarization failure). CONCLUSIONS AND IMPLICATIONS: In silico hiPSC-CMs and hAdultV-CMs exhibit a similar response to selective current blocks. However, overall hiPSC-CMs show greater sensitivity to block, which may facilitate in vitro identification of drug-induced effects. Extrapolation of drug effects from hiPSC-CM to hAdultV-CM and pro-arrhythmic risk assessment can be facilitated by in silico predictions using biophysically-based computational models.

The combination of electric current and copper promotes neuronal differentiation of adipose-derived stem cells.

Damage to the nervous system can be caused by several types of insults, and it always has a great effect on the life of an individual. Due to the limited availability of neural transplants, alternative approaches for neural regeneration must be developed. Stem cells have a great potential to support neuronal regeneration. Human adipose-derived stem cells (hADSCs) have gained increasing interest in the fields of regenerative medicine due to their multilineage potential and easy harvest compared to other stem cells. In this study, we present a growth factor-free method for the differentiation of hADSCs toward neuron-like cells. We investigated the effect of electric current and copper on neuronal differentiation. We analyzed the morphological changes, the mRNA and protein expression levels in the stimulated cells and showed that the combination of current and copper induces stem cell differentiation toward the neuronal lineage with elongation of the cells and the upregulation of neuron-specific genes and proteins. The induction of the neuronal differentiation of hADSCs by electric field and copper may offer a novel approach for stem cell differentiation and may be a useful tool for safe stem cell-based therapeutic applications.

Optical projection tomography as a tool for 3D imaging of hydrogels.

An Optical Projection Tomography (OPT) system was developed and optimized to image 3D tissue engineered products based in hydrogels. We develop pre-reconstruction algorithms to get the best result from the reconstruction procedure, which include correction of the illumination and determination of sample center of rotation (CoR). Existing methods for CoR determination based on the detection of the maximum variance of reconstructed slices failed, so we develop a new CoR search method based in the detection of the variance sharpest local maximum. We show the capabilities of the system to give quantitative information of different types of hydrogels that may be useful in its characterization.

Oxidative stress plays an important role in zoledronic acid-induced autophagy.

Several pre-clinical and clinical studies have demonstrated zoledronic acid (Zol), which regulates the mevalonate pathway, has efficient anti-cancer effects. Zol can also induce autophagy. The aim of this study is to add new understanding to the mechanism of autophagy induction by Zol. LC3B-II, the marker for autophagy was increased by Zol treatment in breast cancer cells. Autophagosomes induced by Zol were visualized and quantified in both transient (pDendra2-hLC3) and stable MCF-7-GFP-LC3 cell lines. Acidic vesicular organelles were quantified using acridine orange. Zol induced a dose and time dependent autophagy. Treatment of Zol increased oxidative stress in MCF-7 cells, which was reversed by GGOH or anti-oxidants. On the other hand, treatment with GGOH or anti-oxidants resulted in decreased levels of LC3B-II. Further, the induced autophagy was irreversible, as the washout of Zol after 2 h or 24 h resulted in similar levels of autophagy, as induced by continuous treatment after 72 h. Thus, it can be summarized that Zol can induce a dose dependent but irreversible autophagy, by its effect on the mevalonate pathway and oxidative stress. This study adds to the understanding of the mechanism of action of Zol, and that it can induce autophagy at clinically relevant shorter exposure times in cancer cells.

Hyaluronan synthase 1 (HAS1) produces a cytokine-and glucose-inducible, CD44-dependent cell surface coat.

Hyaluronan is a ubiquitous glycosaminoglycan involved in embryonic development, inflammation and cancer. In mammals, three hyaluronan synthase isoenzymes (HAS1-3) inserted in the plasma membrane produce hyaluronan directly on cell surface. The mRNA level and enzymatic activity of HAS1 are lower than those of HAS2 and HAS3 in many cells, obscuring the importance of HAS1. Here we demonstrate using immunocytochemistry and transfection of fluorescently tagged HAS1 that its enzymatic activity depends on the ER-Golgi-plasma membrane traffic, like reported for HAS2 and HAS3. When cultured in 5 mM glucose, HAS1-transfected MCF-7 cells show very little cell surface hyaluronan, detected with a fluorescent hyaluronan binding probe. However, a large hyaluronan coat was seen in cells grown in 20 mM glucose and 1 mM glucosamine, or treated with IL-1ß, TNF-α, or TGF-ß. The coats were mostly removed by the presence of hyaluronan hexasaccharides, or Hermes1 antibody, indicating that they depended on the CD44 receptor, which is in a contrast to the coat produced by HAS3, remaining attached to HAS3 itself. The findings suggest that HAS1-dependent coat is induced by inflammatory agents and glycemic stress, mediated by altered presentation of either CD44 or hyaluronan, and can offer a rapid cellular response to injury and inflammation.

Intercellular Ca(2+) wave propagation in human retinal pigment epithelium cells induced by mechanical stimulation.

Ca(2+) signaling is vitally important in cellular physiological processes and various drugs also affect Ca(2+) signaling. Thus, knowledge of Ca(2+) dynamics is important toward understanding cell biology, as well as the development of drug-testing assays. ARPE-19 cells are widely used for modeling human retinal pigment epithelium functions and drug-testing, but intercellular communication has not been assessed in these cells. In this study, we investigated intercellular Ca(2+) communication induced by mechanical stimulation in ARPE-19 cells. An intercellular Ca(2+) wave was induced in ARPE-19 monolayer by point mechanical stimulation of a single cell. Dynamic changes of intracellular Ca(2+) concentration ([Ca(2+)](i)) in the monolayer were tracked with fluorescence microscopy imaging using Ca(2+)-sensitive fluorescent dye fura-2 in presence and absence of extracellular Ca(2+), after depletion of intracellular Ca(2+) stores with thapsigargin, and after application of gap junction blocker α-glycyrrhetinic acid and P2-receptor blocker suramin. Normalized fluorescence values, reflecting amplitude of [Ca(2+)](i) increase, and percentage of responsive cells were calculated to quantitatively characterize Ca(2+) wave propagation. Mechanical stimulation of a single cell within a confluent monolayer of ARPE-19 cells initiated an increase in [Ca(2+)](i), which propagated to neighboring cells in a wave-like manner. Ca(2+) wave propagated to up to 14 cell tiers in control conditions. The absence of extracellular Ca(2+) reduced [Ca(2+)](i) increase in the cells close to the site of mechanical stimulation, whereas the depletion of intracellular Ca(2+) stores with thapsigargin blocked the wave spreading to distant cells. The gap junction blocker α-glycyrrhetinic acid reduced [Ca(2+)](i) increase in the cell tiers close to the site of mechanical stimulation, indicating involvement of gap junctions in Ca(2+) wave propagation. The P2-receptor blocker suramin reduced the percentage of responsive cells participating in Ca(2+) wave spreading beyond the fourth cell tier, showing the necessity of P2-receptors for Ca(2+) wave propagation. In disconnected, i.e., subconfluent, ARPE-19 cell clusters Ca(2+) wave spreading was considerably less efficient compared to that in confluent ARPE-19 monolayer at the same distances. ARPE-19 cells showed repeatable and robust Ca(2+) dynamics after mechanical stimulus. The ARPE-19 cells exhibited two different mechanisms of Ca(2+) wave propagation dependent on the cell location: in the cells close to the site of mechanical stimulation the Ca(2+) wave propagated mainly through gap junctions and required Ca(2+) from both intracellular Ca(2+) stores and extracellular media, while farther away the propagation was more dependent on the purinergic receptors and did not require extracellular Ca(2+). The proposed method could provide a tool to assess the drug-induced changes in intercellular communication in in vitro assays in human retinal pigment epithelial cells.

Multilead measurement system for the time-domain analysis of bioimpedance magnitude.

Bioimpedance measurement applications range from the characterization of organic matter to the monitoring of biological signals and physiological parameters. Occasionally, multiple bioimpedances measured in different locations are combined in order to solve complex problems or produce enhanced physiological measures. The present multilead bioimpedance measurement methods are mainly focused on electrical impedance tomography. Systems designed to suit other multilead applications are lacking. In this study, a novel multilead bioimpedance measurement system was designed. This was particularly aimed at the time-domain analysis of bioimpedance magnitude. Frequency division multiplexing was used to avoid overlapping between excitation signals; undersampling, to reduce the hardware requirements; and power isolated active current sources, to reduce the electrical interactions between leads. These theoretical concepts were implemented on a prototype device. The prototype was tested on equivalent circuits and a saline tank in order to assess excitation signal interferences and electrical interactions between leads. The results showed that the proposed techniques are functional and the system's validity was demonstrated on a real application, multilead impedance pneumography. Potential applications and further improvements were discussed. It was concluded that the novel approach potentially enables accurate and relatively low-power multilead bioimpedance measurements systems.

Impedance spectroscopy in monitoring the maturation of stem cell-derived retinal pigment epithelium.

The development and differentiation of stem cell-derived impermeable retinal pigment epithelium (RPE) with tight junctions (TJs) is a gradual process that is, at confluence, controlled by cell-to-cell contact. The objective of this study was to evaluate the use of electric impedance spectroscopy (EIS) to follow the maturation and development of barrier function in human embryonic stem cell-derived RPE (hESC-RPE). Barrier function was assessed using EIS, permeability measurements, and microscopic inspection in intact cells and following calcium sequestration with ethylene glycol tetraacetic acid (EGTA). The results showed that the cultures with the most mature morphology had the highest impedance and the lowest permeability values. The EIS of samples of high integrity fitted well to the equivalent model of a single RC circuit, whereas the semicircular shape of the Nyquist plots was distorted for samples of lower integrity. EGTA treatment resulted in lower impedance values and changes in the shapes of plots. Our results show that EIS-as a measure of overall maturity and integrity of the epithelium-is useful when evaluating the maturity of cell cultures. It is highly warranted in future transplantation therapies and in in vitro cell culture models in drug development.

A method for suppressing cardiogenic oscillations in impedance pneumography.

The transthoracic electrical impedance signal originates from the cardiac and respiratory functions. In impedance pneumography (IP) the lung function is assessed and the cardiac impedance signal, cardiogenic oscillations (CGOs), is considered an additive noise in the measured signal. In order to accurately determine pulmonary flow parameters from the signal, the CGO needs to be attenuated without distorting the respiratory part of the signal. We assessed the suitability of a filtering technique, originally described by Schuessler et al (1998 Ann. Biomed. Eng. 26 260-7) for an esophageal pressure signal, for CGO attenuation in the IP signal. The technique is based on ensemble averaging the CGO events using the electrocardiogram (ECG) R-wave as the trigger signal. Lung volume is known to affect the CGO waveforms. Therefore we modified the filtering method to produce a lung volume-dependent parametric model of the CGO waveform. A simultaneous recording of ECG, IP and pneumotachograph (PNT) was conducted on 41 healthy, sitting adults. The performance of the proposed method was compared to a low-pass filter and a Savitzky-Golay filter in terms of CGO attenuation and respiratory signal distortion. The method was found to be excellent, exhibiting CGO attenuation of 35.0±12.5 dB (mean±SD) and minimal distortion of the respiratory part of the impedance signal.

Enhanced bone structural analysis through pQCT image preprocessing.

Several factors, including preprocessing of the image, can affect the reliability of pQCT-measured bone traits, such as cortical area and trabecular density. Using repeated scans of four different liquid phantoms and repeated in vivo scans of distal tibiae from 25 subjects, the performance of two novel preprocessing methods, based on the down-sampling of grayscale intensity histogram and the statistical approximation of image data, was compared to 3 x 3 and 5 x 5 median filtering. According to phantom measurements, the signal to noise ratio in the raw pQCT images (XCT 3000) was low ( approximately 20dB) which posed a challenge for preprocessing. Concerning the cortical analysis, the reliability coefficient (R) was 67% for the raw image and increased to 94-97% after preprocessing without apparent preference for any method. Concerning the trabecular density, the R-values were already high ( approximately 99%) in the raw images leaving virtually no room for improvement. However, some coarse structural patterns could be seen in the preprocessed images in contrast to a disperse distribution of density levels in the raw image. In conclusion, preprocessing cannot suppress the high noise level to the extent that the analysis of mean trabecular density is essentially improved, whereas preprocessing can enhance cortical bone analysis and also facilitate coarse structural analyses of the trabecular region.

Cis-urocanic acid suppresses UV-B-induced interleukin-6 and -8 secretion and cytotoxicity in human corneal and conjunctival epithelial cells in vitro.

PURPOSE: Urocanic acid (UCA) is a major ultraviolet (UV)-absorbing endogenous chromophore in the epidermis and is also an efficacious immunosuppressant. The anti-inflammatory and cytoprotective effects of cis-UCA were studied in ocular surface cell cultures exposed to UV-B irradiation. METHODS: Human corneal epithelial cells (HCE-2) and human conjunctival epithelial cells (HCECs) were incubated with 10, 100, 1,000, and 5,000 microg/ml cis-UCA with and without a single UV-B irradiation dose. The concentrations of IL-1beta, IL-6, IL-8, and TNF-alpha in the culture medium and caspase-3 activity in the cell extract sampled were measured by enzyme-linked immunosorbent assay (ELISA). Cell viability was measured by the colorimetric MTT (3-(4,5-dimethyldiazol- 2-yl)-2,5-diphenyltetrazolium bromide) assay. RESULTS: UV-B irradiation multiplied interleukin IL-6 and IL-8 secretion levels in HCE-2 cells and HCECs as analyzed with ELISA. Cell viability as measured by the MTT assay declined by 30%-50% in HCE-2 cells and by 20%-40% in HCECs after UV-B irradiation. Moreover, UV-B increased caspase-3 activity in both cell types as analyzed with ELISA. Treatment with 100 microg/ml cis-UCA completely suppressed IL-6 and IL-8 secretion, decreased caspase-3 activity, and improved cell viability against UV-B irradiation. No significant effects on IL-6 or IL-8 secretion, caspase-3 activity, or viability of the non-irradiated cells were observed with 100 microg/ml cis-UCA in both cell types. The 5,000 microg/ml concentration was toxic. CONCLUSIONS: These findings indicate that cis-UCA may represent a promising anti-inflammatory and cytoprotective treatment option to suppress UV-B-induced inflammation and cellular damage in human corneal and conjunctival epithelial cells.