Lilla Knels

Progression of Parkinson's Disease Pathology Is Reproduced by Intragastric Administration of Rotenone in Mice

In patients with Parkinsons disease (PD), the associated pathology follows a characteristic pattern involving inter alia the enteric nervous system (ENS), the dorsal motor nucleus of the vagus (DMV), the intermediolateral nucleus of the spinal cord and the substantia nigra, providing the basis for the neuropathological staging of the disease. Here we report that intragastrically administered rotenone, a commonly used pesticide that inhibits Complex I of the mitochondrial respiratory chain, is able to reproduce PD pathological staging as found in patients. Our results show that low doses of chronically and intragastrically administered rotenone induce alpha-synuclein accumulation in all the above-mentioned nervous system structures of wild-type mice. Moreover, we also observed inflammation and alpha-synuclein phosphorylation in the ENS and DMV. HPLC analysis showed no rotenone levels in the systemic blood or the central nervous system (detection limit [rotenone]<20 nM) and mitochondrial Complex I measurements showed no systemic Complex I inhibition after 1.5 months of treatment. These alterations are sequential, appearing only in synaptically connected nervous structures, treatment time-dependent and accompanied by inflammatory signs and motor dysfunctions. These results strongly suggest that the local effect of pesticides on the ENS might be sufficient to induce PD-like progression and to reproduce the neuroanatomical and neurochemical features of PD staging. It provides new insight into how environmental factors could trigger PD and suggests a transsynaptic mechanism by which PD might spread throughout the central nervous system.

Promotion of cell adherence and spreading: a novel function of RAGE, the highly selective differentiation marker of human alveolar epithelial type I cells

The receptor for advanced glycation endproducts (RAGE) is expressed under pathological conditions in many tissues and has been assigned many functions. We demonstrate, in normal human lung tissue, the preferential and highly abundant expression of RAGE by quantitative polymerase chain reaction. In addition, RAGE expression, as a specific differentiation marker of alveolar epithelial type I cells (AT I cells), and its localization to the basolateral plasma membrane have been confirmed by means of newly raised monoclonal antibodies. The physiological function of RAGE on AT I cells has previously remained elusive. By using HEK293 cells transfected with cDNA encoding for full-length RAGE, we show that RAGE enhances the adherence of epithelial cells to collagen-coated surfaces and has a striking capacity for inducing cell spreading. The preferential binding of RAGE to collagen has been confirmed by assaying the binding of soluble RAGE to various substrates. RAGE might thus assist AT I cells to acquire a spreading morphology, thereby ensuring effective gas exchange and alveolar stability.

Distribution of Endothelin Receptor Subtypes ETA and ETB in the Rat Kidney

The endothelin (ET) receptor system is markedly involved in the regulation of renal function under both physiological and pathophysiological conditions. The present study determined the detailed cellular localization of both ET receptor subtypes, ETA and ETB, in the vascular and tubular system of the rat kidney by immunofluorescence microscopy. In the vascular system we observed both ETA and ETB receptors in the media of interlobular arteries and afferent and efferent arterioles. In interlobar and arcuate arteries, only ETA receptors were present on vascular smooth muscle cells. ETB receptor immunoreactivity was sparse on endothelial cells of renal arteries, whereas there was strong labeling of peritubular and glomerular capillaries as well as vasa recta endothelium. ETA receptors were evident on glomerular mesangial cells and pericytes of descending vasa recta bundles. In the renal tubular system, ETB receptors were located in epithelial cells of proximal tubules and inner medullary collecting ducts, whereas ETA receptors were found in distal tubules and cortical collecting ducts. Distribution of ETA and ETB receptors in the vascular and tubular system of the rat kidney reported in the present study supports the concept that both ET receptor subtypes cooperate in mediating renal cortical vasoconstriction but exert differential and partially antagonistic effects on renal medullary function. (J Histochem Cytochem 54:1193-1203, 2006)

Imaging of the three-dimensional alveolar structure and the alveolar mechanics of a ventilated and perfused isolated rabbit lung with Fourier domain optical coherence tomography

In this feasibility study, Fourier domain optical coherence tomography (FDOCT) is used for visualizing the 3-D structure of fixated lung parenchyma and to capture real-time cross sectional images of the subpleural alveolar mechanics in a ventilated and perfused isolated rabbit lung. The compact and modular setup of the FDOCT system allows us to image the first 500 microm of subpleural lung parenchyma with a 3-D resolution of 16 x 16 x 8 microm (in air). During mechanical ventilation, real-time cross sectional FDOCT images visualize the inflation and deflation of alveoli and alveolar sacks (acini) in successive images of end-inspiratory and end-expiratory phase. The FDOCT imaging shows the relation of local alveolar mechanics to the setting of tidal volume (VT), peak airway pressure, and positive end-expiratory pressure (PEEP). Application of PEEP leads to persistent recruitment of alveoli and acini in the end-expiratory phase, compared to ventilation without PEEP where alveolar collapse and reinflation are observed. The imaging of alveolar mechanics by FDOCT will help to determine the amount of mechanical stress put on the alveolar walls during tidal ventilation, which is a key factor in understanding the development of ventilator induced lung injury (VILI).

Simultaneous three-dimensional optical coherence tomography and intravital microscopy for imaging subpleural pulmonary alveoli in isolated rabbit lungs

There is a growing interest in analyzing lung mechanics at the level of the alveoli in order to understand stress-related pathogenesis and possibly avoid ventilator associated lung injury. Emerging quantitative models to simulate fluid mechanics and the associated stresses and strains on delicate alveolar walls require realistic quantitative input on alveolar geometry and its dynamics during ventilation. Here, three-dimensional optical coherence tomography (OCT) and conventional intravital microscopy are joined in one setup to investigate the geometric changes of subpleural alveoli during stepwise pressure increase and release in an isolated and perfused rabbit lung model. We describe good qualitative agreement and quantitative correlation between the OCT data and video micrographs. Our main finding is the inflation and deflation of individual alveoli with noticeable hysteresis. Importantly, this three-dimensional geometry data can be extracted and converted into input data for numerical simulations.

Comparative effects of vaporized perfluorohexane and partial liquid ventilation in oleic acid-induced lung injury

Background:It is currently not known whether vaporized perfluorohexane is superior to partial liquid ventilation (PLV) for therapy of acute lung injury. In this study, the authors compared the effects of both therapies in oleic acid–induced lung injury. Methods:Lung injury was induced in 30 anesthetized and mechanically ventilated pigs by means of central venous infusion of oleic acid. Animals were assigned to one of the following groups: (1) control or gas ventilation (GV), (2) 2.5% perfluorohexane vapor, (3) 5% perfluorohexane vapor, (4) 10% perfluorohexane vapor, or (5) PLV with perfluorooctane (30 ml/kg). Two hours after randomization, lungs were recruited and positive end-expiratory pressure was adjusted to obtain minimal elastance. Ventilation was continued during 4 additional hours, when animals were killed for lung histologic examination. Results:Gas exchange and elastance were comparable among vaporized perfluorohexane, PLV, and GV before the open lung approach was used and improved in a similar fashion in all groups after positive end-expiratory pressure was adjusted to optimal elastance (P < 0.05). A similar behavior was observed in functional residual capacity (FRC) in animals treated with vaporized perfluorohexane and GV. Lung resistance improved after recruitment (P < 0.05), but values were higher in the 10% perfluorohexane and PLV groups as compared with GV (P < 0.05). Interestingly, positive end-expiratory pressure values required to obtain minimal elastance were lower with 5% perfluorohexane than with PLV and GV (P < 0.05). In addition, diffuse alveolar damage was significantly lower in the 5% and 10% perfluorohexane vapor groups as compared with PLV and GV (P < 0.05). Conclusions:Although the use of 5% vaporized perfluorohexane permitted the authors to reduce pressures needed to stabilize the lungs and was associated with better histologic findings than were PLV and GV, none of these perfluorocarbon therapies improved gas exchange or lung mechanics as compared with GV.

Three-dimensional Fourier domain optical coherence tomography in vivo imaging of alveolar tissue in the intact thorax using the parietal pleura as a window

In vivo determination of 3-D and dynamic geometries of alveolar structures with adequate resolution is essential for developing numerical models of the lung. A thorax window is prepared in anesthetized rabbits by removal of muscle tissue between the third and fourth rib without harming the parietal pleura. The transparent parietal pleura allows contact-free imaging by intravital microscopy (IVM) and 3-D optical coherence tomography (3-D OCT). We demonstrate that dislocation of the lung surface is small enough to observe identical regions in the expiratory and inspiratory plateau phase, and that OCT in this animal model is suitable for generating 3-D geometry of in vivo lung parenchyma. To our knowledge, we present a novel thorax window preparation technique for 3-D imaging of alveolar dynamics for the first time. The 3-D datasets of the fine structure of the lung beneath the pleura could provide a basis for the development of 3-D numerical models of the lung.

Improved three-dimensional Fourier domain optical coherence tomography by index matching in alveolar structures

Three-dimensional Fourier domain optical coherence tomography (3-D FDOCT) is used to demonstrate that perfusion fixation with a mixture of glutaraldehyde and paraformaldehyde does not alter the geometry of subpleural lung parenchyma in isolated and perfused rabbit lungs. This is confirmed by simultaneous imaging of lung parenchyma with intravital microscopy. To eliminate the diffraction index interfaces between alveolar pockets and walls, we fill the fixed lungs with ethanol by perfusing with gradually increasing concentrations. This bottom-up filling process leaves no remaining air bubbles in the alveolar structures, thus drastically improving the resolution and penetration depth of 3-D FDOCT imaging. We observe an approximately 18% increase in alveolar area after ethanol filling, likely due in large part to elimination of the air/tissue interfaces. 3-D OCT datasets acquired from ethanol-filled lungs allow segmentation of the ethanol-filled structures, which were formerly air-filled, and 3-D reconstruction of larger areas of subpleural alveolar structures. Our innovative process of filling the lungs with ethanol postperfusion fixation thus enables more accurate quantification of alveolar geometries, a critical component of modeling lung function.

Serum-free corneal organ culture medium (SFM) but not conventional minimal essential organ culture medium (MEM) protects human corneal endothelial cells from apoptotic and necrotic cell death

Aim To evaluate the influence of organ culture media on corneal endothelial cell survival. Methods The human corneal endothelial cell line HCEC-12 was cultured in five different media: human corneal endothelial cell (HCEC) growth medium (F99HCEC), standard minimal essential corneal organ culture medium (MEM)+2% fetal calf serum (FCS), MEM+5% FCS, and humanised, endothelial serum-free medium (SFM) (with and without antibiotics). A portion of the cells was treated with 0.5 μmol/l staurosporine and examined for signs of apoptosis by assessing mitochondrial membrane polarisation state (intravital JC-1 staining), by YO-PRO-1 and propidium iodide staining, by determining fragmentation of nuclei by sub-G1 DNA content, by immunocytochemistry for cleaved caspase-3, cleaved caspase-8, Bcl2-associated X protein (Bax) and B-cell lymphoma 2 (Bcl-2), and by western blotting for cleaved caspase-3 and cleaved poly (ADP-ribose) polymerase (PARP). Results The number of apoptotic cells in untreated control cultures was significantly higher in MEM compared with F99HCEC and SFM. Staurosporine treatment induced apoptosis in all tested cultures to varying degrees. Cells cultured in MEM showed stronger staining for cleaved caspase-3, cleaved caspase-8, Bax, Bcl-2 and cleaved PARP, increased sub-G1 DNA content, more propidium iodide- and YO-PRO-1-positive cells, and more mitochondria with depolarised membranes. All parameters were significantly higher in MEM compared with F99HCEC and SFM. SFM cultures were significantly less susceptible to cell stress. Conclusion SFM is superior to MEM in promoting HCEC survival.

Blue light stress in retinal neuronal (R28) cells is dependent on wavelength range and irradiance

The aim of our study was to elucidate the role of wavelength and irradiance in blue light retinal damage. We investigated the impact of blue light emitted from light‐emitting diode (LED) modules with peaks at either 411 nm (half bandwidth 17 nm) or 470 nm (half bandwidth 25 nm) at defined irradiances of 0.6, 1.5 and 4.5 W/m2 for 411 nm and 4.5 W/m2 for 470 nm on retinal neuronal (R28) cells in vitro. We observed a reduction in metabolic activity and transmembrane potential of mitochondria when cells were irradiated at 411 nm at higher irradiances. Furthermore, production of mitochondrial superoxide radicals increased significantly when cells were irradiated with 411 nm light at 4.5 W/m2. In addition, such irradiation caused an activation of the antioxidative glutathion system. Using vital staining, flow cytometry and western blotting, we were able to show that apoptosis only took place when cells were exposed to 411 nm blue light at higher irradiances; necrosis was not observed. Enhanced caspase‐3 cleavage product levels confirmed that this effect was dependent on light irradiance. Significant alterations of the above‐mentioned parameters were not observed when cells were irradiated with 471 nm light despite a high irradiance of 4.5 W/m2, indicating that the cytotoxic effect of blue light is highly dependent on wavelength. The observed phenomena in R28 cells at 411 nm (4.5 W/m2) point to an apoptosis pathway elicited by direct mitochondrial damage and increased oxidative stress. Thus, light of 411 nm should act via impairment of mitochondrial function by compromising the metabolic situation of these retinal neuronal cells.