Familial congenital heart disease, progressive atrioventricular block and the cardiac homeobox transcription factor gene NKX2.5: identification of a novel mutation.
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Adult↗
Biomedical subjects
K König
Publications and source records attributed to K König.
Influence of nanoencapsulation on human skin transport of flufenamic acid.
The effect of the inclusion of flufenamic acid in poly(lactide-co-glycolide) nanoparticles on the transport of flufenamic acid into excised human skin was investigated. Penetration and permeation data were acquired using two different in vitro test systems: the Saarbrucken penetration model, where the skin acts as its own receptor medium, and the Franz diffusion cell, where the receptor medium is a buffer solution. For the stratum corneum, no differences were found between nanoencapsulated and free drug. Drug accumulation in the deeper skin layers and drug transport across human epidermis were slightly delayed for the nanoencapsulated drug compared to the free drug after shorter incubation times (<12 h). In contrast, after longer incubation times (>12 h), the nanoencapsulated drug showed a statistically significantly enhanced transport and accumulation (p < 0.05). Additionally, nanoencapsulated flufenamic acid was visualized by multiphoton fluorescence microscopy. Particles were found homogeneously distributed on the skin surface and within the dermatoglyphs, but no nanoparticles were detected within or between the corneocytes.
Administration, Cutaneous↗
Fluorescence lifetime images and correlation spectra obtained by multidimensional time-correlated single photon counting.
Multidimensional time-correlated single photon counting (TCSPC) is based on the excitation of the sample by a high-repetition rate laser and the detection of single photons of the fluorescence signal in several detection channels. Each photon is characterized by its arrival time in the laser period, its detection channel number, and several additional variables such as the coordinates of an image area, or the time from the start of the experiment. Combined with a confocal or two-photon laser scanning microscope and a pulsed laser, multidimensional TCSPC makes a fluorescence lifetime technique with multiwavelength capability, near-ideal counting efficiency, and the capability to resolve multiexponential decay functions. We show that the same technique and the same hardware can be used for precision fluorescence decay analysis and fluorescence correlation spectroscopy (FCS) in selected spots of a sample.
Cell Line↗
Age-dependence of cerebrospinal parameters.
OBJECTIVE: The records of 159 patients were reviewed who routinely underwent a classical volume-pressure testing procedure due to suspected cerebrospinal fluid circulatory disorders. METHODS: Regression analyses were carried out to explain the cerebrospinal parameters' relationships to age. Least median squares (LMS) algorithms achieved robust estimation of simple linear model parameters. Also, method of weighted regression was used because of unequal variances in the observations. RESULTS: Cerebrospinal elastance and resistance to outflow revealed significant LMS regressions: y = 0.171x + 7.460 (n = 159, p < 0.0001) and y = 0.096x + 1.871 (n = 97, p < 0.0001), respectively. Similarly, weighting data resulted in models such as y = 0.151x + 6.830 (p < 0.0001) for elastance and y = 0.087x + 1.730 (p < 0.0001) for resistance estimates. The intracranial pressure at rest showed no age-related dependency. Both clear clinical and morphological signs were found in 20% of patients. CONCLUSION: Expecting no time-variant properties we surprisingly found a significant linear relationship in cerebrospinal parameters and age. Thus, parameter magnitudes are not likely dominated by a pathological process only but also determined by temporal system alterations.
Adolescent↗
Multiphoton autofluorescence imaging of intratissue elastic fibers.
Multiphoton induced blue/green autofluorescence by near infrared femtosecond laser pulses has been used to selectively image intratissue elastic fibers in native and tissue engineered (TE) viable heart valves without any invasive tissue removal, embedding, fixation, and staining. Elastic fibers could be clearly distinguished from collagenous structures which emit ultraviolet/violet radiation when excited with intense ultrashort pulses due to second harmonic generation. Deep-tissue three-dimensional imaging of elastic fibers with submicron spatial resolution was performed by optical sectioning of heart valves using a multiphoton laser scanning microscope in connection with a tunable 80 MHz femtosecond laser source. The technology was used to diagnose extracellular matrix structures and cell resettlement of TE heart valves prior implantation. This novel non-invasive method opens the general possibility of high-resolution in situ imaging of elastic fibers, collagen structures and intracellular organelles in living intact tissues without staining.
Animals↗
Fluorescence lifetime imaging by time-correlated single-photon counting.
We present a time-correlated single photon counting (TCPSC) technique that allows time-resolved multi-wavelength imaging in conjunction with a laser scanning microscope and a pulsed excitation source. The technique is based on a four-dimensional histogramming process that records the photon density over the time of the fluorescence decay, the x-y coordinates of the scanning area, and the wavelength. The histogramming process avoids any time gating or wavelength scanning and, therefore, yields a near-perfect counting efficiency. The time resolution is limited only by the transit time spread of the detector. The technique can be used with almost any confocal or two-photon laser scanning microscope and works at any scanning rate. We demonstrate the application to samples stained with several dyes and to CFP-YFP FRET.
Animals↗
Comparative study of cellular and extracellular matrix composition of native and tissue engineered heart valves.
Tissue engineering of heart valves utilizes biodegradable or metabolizable scaffolds for remodeling by seeded autologous cells. The aim of this study was to determine and compare extracellular matrix (ECM) formations, cellular phenotypes and cell location of native and tissue engineered (TE) valve leaflets. Ovine carotid arteries, ovine and porcine hearts were obtained from slaughterhouses. Cells were isolated from carotid arteries and dissected ovine, porcine and TE leaflets. TE constructs were fabricated from decellularized porcine pulmonary valves, seeded ovine arterial cells and subsequent 16 days dynamic in vitro culture using a pulsatile bioreactor. Native and TE valves were studied by histology (hematoxylin-eosin, resorcin-fuchsin, Movat pentachrome), NIR femtosecond multiphoton laser scanning microscopy and scanning electron microscopy (SEM). Cells of native and TE tissues were identified and localized by immunohistochemistry. Arterial, valvular and re-isolated TE-construct cells were processed for immunocytochemistry and Western blotting. ECM analysis and SEM revealed characteristical and comparable structures in native and TE leaflets. Most cells in native leaflets stained strongly positive for vimentin. Cells positive to alpha-smooth muscle actin (alpha-SMA), myosin and calponin were only found at the ventricular (inflow) side of ovine aortic and porcine pulmonary valve leaflets. Cells from TE constructs had a strong expression of vimentin, alpha-SMA, myosin, calponin and h-caldesmon throughout the entire leaflet. Comparable ECM formation and endothelial cell lining of native and TE leaflets could be demonstrated. However, immunostaining revealed significant differences between valvular cell phenotypes of native and TE leaflets. These results may be essential for further cardiovascular tissue engineering efforts.
Animals↗
[The angle stable locking-proximal-humerus-plate (LPHP) for proximal humeral fractures using a small anterior-lateral-deltoid-splitting-approach - technique and first results].
The Locking-Proximal-Humerus-Plate (LPHP) was slided from proximally to distally through a small anterior-lateral-deltoid-splitting-approach following a closed reduction. Additional tension band wiring for the rotator cuff was fixed at the plate after screw insertion. The prospective study included 35 patients (age median: 63 years, range: 33-92; male: 13, female: 22) from 08/01-05/02. The follow-up period was set to 3 months postoperatively, whereas 29 patients were able to attend. The patient-group with 2-part fractures (n = 8) showed an average Constant score of 77.6 points (+/- 10.7, "good"), the patients with 3-part fractures (n = 16) an average score of 75.1 points (+/- 14.4, "good") and the patients with 4-part fractures (n = 5) an average score of 64.8 points (+/- 10. 4, "moderate"). Specific, approach-related problems were not observed. Almost all fractures revealed bony union during the first 3 months and only one loss of reduction was found. A screw loosening in 2 cases and a break of the plate in 3 cases were seen as implant related problems, and furthermore in 5 cases the screws placed in the humeral head were too long. Only one early re-osteosynthesis with the same plate was necessary due to an initial non-correct reposition. As demonstrated by these good short-term results, this technique seems to be a suitable procedure for displaced humeral head fractures.
Adult↗
Assessment of cerebral blood flow by means of blood-flow-volume measurement.
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Blood Flow Velocity↗
Impact of decellularization of xenogeneic tissue on extracellular matrix integrity for tissue engineering of heart valves.
The multidisciplinary research of tissue engineering utilizes biodegradable or decellularized scaffolds with autologous cell seeding. Objective of this study was to investigate the impact of different decellularization protocols on extracellular matrix integrity of xenogeneic tissue by means of multiphoton femtosecond laser scanning microscopy, biochemical and histological analysis. Pulmonary valves were dissected from porcine hearts and placed in a solution of trypsin-EDTA and incubated at 37 degrees C for either 5, 8, or 24 h, followed by a 24 h PBS washing. Native and decellularized valves were processed for histology, DNA, cell proliferation, matrix proteins and biomechanical testing. Multiphoton NIR laser microscopy has been applied for high-resolution 3D imaging of collagen and elastin. Distinct differences in several ECM components following decellularization time were observed. Total GAG contents decreased in a time-dependent manner, with o-sulfated GAGs being more susceptible to degradation than n-sulfated GAGs. Efficiency of insoluble collagen extraction increased proportionally with decellularization time, suggesting ECM-integrity may be compromised with prolonged incubation. Biomechanical testing revealed a gradual weakening of mechanical strength with increased decellularization time. The enzymatic decellularization process of heart valves revealed a time-dependent loss of cells, ECM components and biomechanical strength. In order to avoid any immune response a thorough decellularization of 24 h remains mandatory.
Animals↗
Unconventional myosins of the plant-specific class VIII: endocytosis, cytokinesis, plasmodesmata/pit-fields, and cell-to-cell coupling.
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Actins↗
Acupuncture in the treatment of psychogenic erectile dysfunction: first results of a prospective randomized placebo-controlled study.
In a prospective study, we investigated the potentially curative effect of acupuncture in patients with psychogenic erectile dysfunction (pED). A total of 22 patients with pED were randomized into two groups. They were either treated with acupuncture specific against ED (treatment group) or acupuncture specific against headache (placebo group). Nonresponders of the placebo group were crossed over to the treatment group. Prior to acupuncture, serum sexual hormone levels, IIEF score, nocturnal penile tumescence testing for three nights (Rigiscan) and the erectile response to 50 mg sildenafil were evaluated. Out of 21 patients, 20 completed the study, including 10 patients after crossover. A satisfactory response was achieved in 68.4% of the treatment group and in 9% of the placebo group (P=0.0017). Another 21.05% of the patients had improved erections, that is, sufficient rigidity under simultaneous treatment with 50 gm sildenafil. The results of our pilot study indicate that acupuncture can be an effective treatment option in more than two-thirds of patients with psychogenic erectile dysfunction.
Acupuncture Therapy↗
Femtosecond near-infrared laser pulses elicit generation of reactive oxygen species in mammalian cells leading to apoptosis-like death.
Two-photon excitation-based near-infrared (NIR) laser scanning microscopy is currently emerging as a new and versatile alternative to conventional confocal laser scanning microscopy, particularly for vital cell imaging in life sciences. Although this innovative microscopy has several advantages such as highly localized excitation, higher penetration depth, reduced photobleaching and photodamage, and improved signal to noise ratio, it has, however, recently been evidenced that high-power NIR laser irradiation can drastically inhibit cell division and induce cell death. In the present study we have investigated the cellular responses of unlabeled rat kangaroo kidney epithelium (PtK2) cells to NIR femtosecond laser irradiation. We demonstrate that NIR 170-fs laser pulses operating at 80-MHz pulse repetition frequency and at mean power of > or = 7 mW evoke generation of reactive oxygen species (ROS) such as H2O2 that can be visualized in situ by standard in vivo cytochemical analysis using Ni-3,3'-diaminobenzidine (Ni-DAB) as well as with a recently developed fluorescent probe Jenchrom px blue. The formation of the Ni-DAB reaction product as well as that of Jenchrom was relatively more pronounced when irradiated cells were incubated in alkaline solution (pH 8) than in those incubated in acidic solution (pH 6), suggesting peroxisomal localization of these reaction products. Two-photon time-lapse imaging of the internalization of the cell impermeate fluorescent dye propidium iodide revealed that the integrity of the plasma membrane of NIR irradiated cells is drastically compromised. Visualization of the nuclei with DNA-specific fluorescent probes such as 4',6-diamidino-2-phenylindole 24 h postirradiation further provided tangible evidence that the nuclei of these cells undergo several deformations and eventual fragmentation. That these NIR irradiated cells die by apoptosis has been established by in situ detection of DNA strand breaks using the terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling method. Because the reactive oxygen species such as H2O2 and OH* can cause noxious effects such as cell membrane injury by peroxidation of polyunsaturated lipids and proteins and oxidative phosphorylation, and alterations of ATP-dependent Ca2+ pumps, these ROS are likely to contribute to drastic cytological alterations observed in this study following NIR irradiation. Taken together, we have established that NIR laser irradiations at mean power > or = 7 mW delivered at pulse duration time of 170 fs generally used in two- and multiphoton microscopes cause oxidative stress (1) evoking production of ROS, (2) resulting in membrane barrier dysfunction, (3) inducing structural deformations and fragmentation of the nuclei as well as DNA strand breaks, (4) leading to cell death by apoptosis.
Animals↗
Femtosecond near-infrared lasers as a novel tool for non-invasive real-time high-resolution time-lapse imaging of chloroplast division in living bundle sheath cells of Arabidopsis.
Non-linear excitation of fluorophores by contemporaneous absorption of two or more near-infrared (NIR) photons following diffraction-limited focusing with high-numerical-aperture objectives circumvents out-of-focus fluorescence (without a confocal pinhole) and spatially limits photobleaching and photodamage to the minute sub-femtoliter focal volume. This is in contrast to optical events in conventional confocal imaging systems using ultraviolet (UV) or visible laser sources wherein the entire sectors of the specimen above and below the plane of focus experience massive photostress and photodamage. In addition, NIR wavelengths penetrate deeper into the highly scattering environs of plant tissue than UV and visible wavelengths. We delineate a novel non-invasive technique using NIR femtosecond laser pulses at lambda = 740, 760, 780, and 800 nm for induction of chlorophyll fluorescence by the two-photon effect as well as for intra-tissue time-lapse vital three-dimensional imaging of fundamental events of chloroplast division in deeply seated bundle sheath cells of Arabidopsis thaliana (L.) Heynh. leaves. Our findings establish that (i) mature bundle sheath chloroplasts are indeed capable of division, (ii) the dividing chloroplasts assume a distinct constricted/dumbbell-shaped profile with an average lifespan of 20-25 min, (iii) the complete division of the pre-existing chloroplasts occurs within 50 min, and (iv) the two derivative daughter chloroplasts are invariably unequal in size. This novel NIR-laser-based technique has any number of potential applications, including (i) non-invasive intra-vital imaging of molecular and ion dynamics, (ii) non-destructive screening of mutants impaired in photosynthesis, (iii) diagnosis of physiological states of plants and (iv) bio-optical taxonomy.
Arabidopsis↗
Artificial elevation of brain tissue glycerol by administration of a glycerol-containing agent. Case report.
In recent years the development of secondary brain damage and derangement of neurochemical parameters after severe head injury has been monitored using microdialysis. Provided the blood-brain barrier is intact, glycerol is regarded as a potential marker for membrane phospholipid degradation. The authors report a case in which marked elevation of interstitial glycerol was induced after exogenous administration of a glycerol-containing agent. A 25-year-old man was injured in a motorcycle accident and was admitted to the authors' institution with a unilateral dilated and fixed pupil and a Glasgow Coma Scale score of 3. Computerized tomography scans revealed a large subdural hematoma on the left side, subsequent midline shift, and generalized edema. Emergency craniotomy was performed for evacuation of the hematoma. The patient was prepared for multisensory monitoring and a microdialysis catheter was inserted into his left frontal lobe. After a routine enema containing 85% glycerol had been administered, the authors measured a marked increase in glycerol in the dialysate. This occurred while the patient was in as stable a condition as could be expected given the circumstances. The increase in interstitial glycerol in the injured tissue was most likely due to an impaired blood-brain barrier. Thus, the interstitial glycerol concentration had been corrupted by exogenous glycerol, and the marker properties of glycerol in this case became questionable. Consequently, administration of glycerol, which is frequently found in various infusions and emulsions, can promote secondary brain damage by adversely shifting osmotic gradients.
Adult↗
Femtosecond near-infrared laser pulse induced strand breaks in mammalian cells.
Multiphoton laser scanning microscopy (MPLSM) is based on non-resonant simultaneous absorption of two or three near infrared (NIR) photons by a fluorophore in the subfemtoliter focal volume of a high numerical aperture (N.A. 1.3) objective. The higher penetration depth of NIR radiation enables optical sectioning across thick biological specimens and because of the absence of efficient single photon absorbers in the NIR spectral region of 700 to 1200 nm there is hardly any out-of-focus photodamage and photobleaching. Recent in vitro studies (14) have demonstrated that irradiation of supercoiled plasmid DNA with intense multiphoton NIR 810 nm of 140 fs pulse width, 76 MHz pulse repetition rate results in single strand breaks as a result of simultaneous absorption of three or more photons. Herein, we have investigated the influence of 800 nm NIR 170 fs laser pulses, 80 MHz pulse repetition frequency at mean powers of 2 to 20 mW on nuclear DNA of unlabelled PtK2 cells. In situ TdT-mediated dUTP-nick end labelling (TUNEL) revealed that cells exposed to the NIR irradiation above > or =5 mW mean laser power alone contained TUNEL-positive nuclei. The intensity of TUNEL fluorescence was relatively higher at increased mean NIR laser power. These results provide evidence that DNA strand breaks also occur in vivo when mammalian cells are exposed to high average power > or =5 mW NIR irradiation during MPLSM possibly due to multiphoton absorption process. Because intense DNA fragmentation is one of the hall marks of programmed cell death it is hypothesised that NIR induced cell death is by apoptosis.
Animals↗
Robert Feulgen Prize Lecture. Laser tweezers and multiphoton microscopes in life sciences.
Near infrared (NIR) laser microscopy enables optical micromanipulation, piconewton force determination, and sensitive fluorescence studies by laser tweezers. Otherwise, fluorescence images with high spatial and temporal resolution of living cells and tissues can be obtained via non-resonant fluorophore excitation with multiphoton NIR laser scanning microscopes. Furthermore, NIR femtosecond laser pulses at TW/cm2 intensities can be used to realize non-invasive contact-free surgery of nanometer-sized structures within living cells and tissues. Applications of these novel versatile NIR laser-based tools for the determination of motility forces, coenzyme and chlorophyll imaging, three-dimensional multigene detection, non-invasive optical sectioning of tissues ("optical biopsy"), functional protein imaging, and nanosurgery of chromosomes are described.
Awards and Prizes↗
Multiplex FISH and three-dimensional DNA imaging with near infrared femtosecond laser pulses.
We report on a novel technology for multicolor gene and chromosome detection as well as for three-dimensional (3D) DNA imaging by multiphoton excitation of multiple FISH fluorophores and DNA stains. Near infrared femtosecond laser pulses at 770 nm were used to simultaneously excite the visible fluorescence of a wide range of FISH fluorophores, such as FITC, DAC, Cy3, Cy5, Cy5.5, rhodamine, spectrum aqua, spectrum green, spectrum orange, Jenfluor, and Texas red as well as of DNA/chromosome stains, for example Hoechst 33342, DAPI, SYBR green, propidium iodide, ethidium homodimer, and Giemsa. In addition to the advantage of using only one excitation wavelength for a variety of fluorophores, multiphoton excitation provided the intrinsic possibility of 3D fluorescence imaging. The technology has been used in human genetics for the diagnosis of numerical chromosome aberrations and microdeletions. In particular, multicolor 3D images of the intranuclear localization of FISH-labeled chromosome territories in interphase nuclei of amniotic fluid cells have been obtained. Using the high light penetration depth at 770 nm, optical sectioning of Hoechst 33342-labeled DNA within living culture cells and within tissue of living tumor-bearing mice was performed.
Adult↗