Bernhard R. Brehm

Noninvasive imaging of intracellular lipid metabolism in macrophages by Raman microscopy in combination with stable isotopic labeling.

Monocyte-derived macrophages play a key role in atherogenesis because their transformation into foam cells is responsible for deposition of lipids in plaques within arterial walls. The appearance of cytosolic lipid droplets is a hallmark of macrophage foam cell formation, and the molecular basics involved in this process are not well understood. Of particular interest is the intracellular fate of different individual lipid species, such as fatty acids or cholesterol. Here, we utilize Raman microscopy to image the metabolism of such lipids and to trace their subsequent storage patterns. The combination of microscopic information with Raman spectroscopy provides a powerful molecular imaging method, which allows visualization at the diffraction limit of the employed laser light and biochemical characterization through associated spectral information. In order to distinguish the molecules of interest from other naturally occurring lipids spectroscopically, deuterium labels were introduced. Intracellular distribution and metabolic changes were observed for serum albumin-complexed palmitic and oleic acid and cholesterol and quantitatively evaluated by monitoring the increase in CD scattering intensities at 0.5, 1, 3, 6, 24, 30, and 36 h. This approach may also allow for investigating the cellular trafficking of other molecules, such as nutrients, metabolites, and drugs.

Characterization of atherosclerotic plaque depositions by Raman and FTIR imaging

Spectroscopy-based imaging techniques can provide useful biochemical information about tissue samples. Here, we employ Raman and Fourier transform infrared (IR) imaging to characterize composition and constitution of atherosclerotic plaques of rabbits, fed with a high cholesterol diet. The results were compared with conventional light microscopy after staining with hematoxylin eosin, and elastica van Gieson. The spectral unmixing algorithm vertex component analysis was applied for data analysis and image reconstruction. IR microscopy allowed for differentiation between lipids and proteins in plaques of full aortic cross sections. Raman microscopy further discriminated cholesterol esters, cholesterol and triglycerides. FTIR and Raman images were recorded at a resolution near 20 micrometer per pixel for a large field of view. High resolution Raman images at 1 micrometer per pixel revealed structural details at selected regions of interest. The intima-media and the lipid-protein ratio were determined in five specimens for quantitation. These results correlate well with histopathology. The described method is a promising tool for easy and fast molecular imaging of atherosclerosis.

In Vivo Characterization of Atherosclerotic Plaque Depositions by Raman-Probe Spectroscopy and in Vitro Coherent Anti-Stokes Raman Scattering Microscopic Imaging on a Rabbit Model

Visualization as well as characterization of inner arterial plaque depositions is of vital diagnostic interest, especially for the early recognition of vulnerable plaques. Established clinical techniques provide valuable visual information but cannot deliver information about the chemical composition of individual plaques. Here, we employ Raman-probe spectroscopy to characterize the plaque compositions of arterial walls on a rabbit model in vivo, using a miniaturized filtered probe with one excitation and 12 collection fibers integrated in a 1 mm sleeve. Rabbits were treated with a cholesterol-enriched diet. The methodology can improve the efficiency of animal experiments and shows great potential for applications in cardiovascular research. In order to further characterize the plaque depositions visually, coherent anti-Stokes Raman scattering (CARS) microscopy images have been acquired and are compared with the Raman-probe results.

Expanding Multimodal Microscopy by High Spectral Resolution Coherent Anti-Stokes Raman Scattering Imaging for Clinical Disease Diagnostics

Over the past years fast label-free nonlinear imaging modalities providing molecular contrast of endogenous disease markers with subcellular spatial resolution have been emerged. However, applications of these imaging modalities in clinical settings are still at the very beginning. This is because single nonlinear imaging modalities such as second-harmonic generation (SHG) and two-photon excited fluorescence (TPEF) have only limited value for diagnosing diseases due to the small number of endogenous markers. Coherent anti-Stokes Raman scattering (CARS) microscopy on the other hand can potentially be added to SHG and TPEF to visualize a much broader range of marker molecules. However, CARS requires a second synchronized laser source and the detection of a certain wavenumber range of the vibrational spectrum to differentiate multiple molecules, which results in increased experimental complexity and often inefficient excitation of SHG and TPEF signals. Here we report the application of a novel near-infrared (NIR) fiber laser of 1 MHz repetition rate, 65 ps pulse duration, and 1 cm(-1) spectral resolution to realize an efficient but experimentally simple SGH/TPEF/multiplex CARS multimodal imaging approach for a label-free characterization of composition of complex tissue samples. This is demonstrated for arterial tissue specimens demonstrating differentiation of elastic fibers, triglycerides, collagen, myelin, cellular cytoplasm, and lipid droplets by analyzing the CARS spectra within the C-H stretching region only. A novel image analysis approach for multispectral CARS data based on colocalization allows correlating spectrally distinct pixels to morphologic structures. Transfer of this highly precise but compact and simple to use imaging approach into clinical settings is expected in the near future.

Association of waist circumference, traditional cardiovascular risk factors, and stromal‐derived factor‐1 in adolescents

Background: Overweight and the metabolic syndrome (MS) represent dramatically increasing problems in children and adolescents. Waist circumference (WC) is an important factor to determine MS. So far, WC is a predictor of blood pressure, high‐density lipoprotein (HDL), insulin concentration, and visceral fat in adolescents. We investigated whether WC and body mass index standard deviation score (BMI‐SDS) are predictors of adiponectin, stromal‐derived factor (SDF‐1), and soluble E‐selectin (sE‐selectin) as parameters for beginning insulin resistance and endothelial damage.

Characterization of collagen and cholesterol deposition in atherosclerotic arterial tissue using non‐linear microscopy

Atherosclerosis is characterized by the accumulation of lipids within the arterial wall and is commonly diagnosed using standard histology. Non-linear microscopy represents a possible label-free alternative to standard diagnostic methods for imaging various tissue components. Here we employ SHG and CARS microscopy for imaging thin cross-sections of atherosclerotic arterial tissue, demonstrating that both cholesterol deposition in the lumen and collagen in the normal arterial wall can be imaged and discriminated using SHG and CARS microscopy. A simultaneous detection of both forward and backward scattered SHG signals allows distinguishing collagen fibres from cholesterol. Further analysis, based on image pattern evaluation algorithms, is used to characterize collagen organization in the healthy arterial wall against collagen found within plaques. Different values of fibre mean size, distribution and anisotropy are calculated for lumen and media prospectively allowing for automated classification of atherosclerotic lesions. The presented method represents a promising diagnostic tool for evaluating atherosclerotic tissue.

A compact microscope setup for multimodal nonlinear imaging in clinics and its application to disease diagnostics

The past years have seen increasing interest in nonlinear optical microscopic imaging approaches for the investigation of diseases due to the methods unique capabilities of deep tissue penetration, 3D sectioning and molecular contrast. Its application in clinical routine diagnostics, however, is hampered by large and costly equipment requiring trained staff and regular maintenance, hence it has not yet matured to a reliable tool for application in clinics. In this contribution implementing a novel compact fiber laser system into a tailored designed laser scanning microscope results in a small footprint easy to use multimodal imaging platform enabling simultaneously highly efficient generation and acquisition of second harmonic generation (SHG), two-photon excited fluorescence (TPEF) as well as coherent anti-Stokes Raman scattering (CARS) signals with optimized CARS contrast for lipid imaging for label-free investigation of tissue samples. The instrument combining a laser source and a microscope features a unique combination of the highest NIR transmission and a fourfold enlarged field of view suited for investigating large tissue specimens. Despite its small size and turnkey operation rendering daily alignment dispensable the system provides the highest flexibility, an imaging speed of 1 megapixel per second and diffraction limited spatial resolution. This is illustrated by imaging samples of squamous cell carcinoma of the head and neck (HNSCC) and an animal model of atherosclerosis allowing for a complete characterization of the tissue composition and morphology, i.e. the tissues morphochemistry. Highly valuable information for clinical diagnostics, e.g. monitoring the disease progression at the cellular level with molecular specificity, can be retrieved. Future combination with microscopic probes for in vivo imaging or even implementation in endoscopes will allow for in vivo grading of HNSCC and characterization of plaque deposits towards the detection of high risk plaques.

Extra cellular matrix remodelling after heterotopic rat heart transplantation: gene expression profiling and involvement of ED-A+ fibronectin, alpha-smooth muscle actin and B+ tenascin-C in chronic cardiac allograft rejection

Chronic cardiac rejection is represented by cardiac allograft vasculopathy (CAV) and cardiac interstitial fibrosis (CIF) known to cause severe complications. These processes are accompanied by remarkable changes in the cardiac extra cellular matrix (cECM). The aim of our study was to analyse the cECM remodelling in chronic rejection and to elucidate a potential role of ED-A domain containing fibronectin (ED-A+ Fn), alpha smooth muscle actin (ASMA) and B domain containing tenascin-C (B+ Tn-C). A model of chronic rejection after heterotopic rat heart transplantation was used. Allografts, recipient and control hearts were subjected to histological assessment of rejection grade, to real-time PCR based analysis of 84 genes of ECM and adhesion molecules and to immunofluorescence labelling procedures, including ED-A+ Fn, ASMA and B+ Tn-C antibodies. Histological analysis revealed different grades of chronic rejection. By gene expression analysis, a relevant up-regulation of the majority of ECM genes in association with chronic rejection could be shown. For 8 genes, there was a relevant up-regulation in allografts as well as in the corresponding recipient hearts. Association of ASMA positive cells with the grade of chronic rejection could be proven. In CAV and also in CIF there were extensive co-depositions of ED-A+ Fn, ASMA and B+ Tn-C. In conclusion, chronic cardiac allograft rejection is associated with a cECM remodelling. ASMA protein deposition in CAV, and CIF is a valuable marker to detect chronic rejection. Interactions of VSMCs and Fibro-/Myofibroblasts with ED-A+ Fn and B+ Tn-C might functionally contribute to the development of chronic cardiac rejection.

Changes in extra cellular matrix remodelling and re-expression of fibronectin and tenascin-C splicing variants in human myocardial tissue of the right atrial auricle: implications for a targeted therapy of cardiovascular diseases using human SIP format antibodies

Cardiovascular diseases are accompanied by changes in the extracellular matrix (ECM) including the re-expression of fibronectin and tenascin-C splicing variants. Using human recombinant small immunoprotein (SIP) format antibodies, a molecular targeting of these proteins is of therapeutic interest. Tissue samples of the right atrial auricle from patients with coronary artery disease and valvular heart disease were analysed by PCR based ECM gene expression profiling. Moreover, the re-expression of fibronectin and tenascin-C splicing variants was investigated by immunofluoerescence labelling. We demonstrated changes in ECM gene expression depending on histological damage or underlying cardiac disease. An increased expression of fibronectin and tenascin-C mRNA in association to histological damage and in valvular heart disease compared to coronary artery disease could be shown. There was a distinct re-expression of ED-A containing fibronectin and A1 domain containing tenascin-C detectable with human recombinant SIP format antibodies in diseased myocardium. ED-A containing fibronectin showed a clear vessel positivity. For A1 domain containing tenascin-C, there was a particular positivity in areas of interstitial and perivascular fibrosis. Right atrial myocardial tissue is a valuable model to investigate cardiac ECM remodelling. Human recombinant SIP format antibodies usable for an antibody-mediated targeted delivery of drugs might offer completely new therapeutic options in cardiac diseases.

First use of a novel plug-and-play percutaneous circulatory assist device for high-risk coronary angioplasty

Objectives: Novel circulatory assist devices provide hemodynamic stability in high risk coronary interventions. They ensure sufficient organ perfusion during transfer in case of procedural failure or cardiogenic arrest. We describe the first human use of a novel plug-and-play circulatory assist device for high risk coronary angioplasty. Methods: An 84 year old lady suffered syncope with complex fracture of the left forearm. Her syncope was related to a subtotal stenosis of the left main coronary artery associated with an acute myocardial infarction. Additional risk factors were previous cardiac surgery, pulmonary disease, diabetes mellitus, and renal insufficiency. Coronary angiography revealed stenosis of both coronary ostia. The emergency assist device LIFEBRIDGE was connected with the patients circulation by percutaneous cannulation (15F and 17F) of the femoral artery and vein. Results: Stent implantation was performed in both coronary ostia by Judkins technique. The cannulas were removed two hours after the intervention by local compression. Osteosynthesis of the left radius and ulna was performed five days later under regional anesthesia. The patient was discharged without any complains on day 10. Conclusion: This case illustrates the safe and easy use of a novel plug-and-play percutaneous circulatory assist device for high risk interventions. It may be recommended for use in emergency situations.