The Cytoscan (TM) model E-II, a new reflectance microscope for intravital microscopy: Comparison with the standard fluorescence method

The Cytoscan(TM) Model E-II (Cytometrics Inc., Philadelphia, Pa., USA) is a newly developed instrument which functions as an intravital microscope and is small and easily portable. Through the use of orthogonal polarization spectral (OPS) imaging, the Cytoscan Model E-II delivers images of the microcirculation which are comparable to those achieved with intravital fluorescence videomicroscopy (IFM), but without the use of fluorescent dyes. The purpose of this study was to validate the Cytoscan Model E-II instrument against IFM. The experiments were carried out on striated muscle in the dorsal skinfold chamber of the awake Syrian hamster. The following parameters were measured in identical regions of interest in the same animal under baseline conditions and 0.5 and 2 h after a 4-hour period of pressure-induced ischemia: arteriolar diameter, venular diameter and venular red blood cell velocity. Bland-Altman plots showed good agreement between the two techniques for venular red blood cell velocity. As expected, arteriolar and venular diameters as measured by the Cytoscan were on average 5 mum smaller than the values from IFM, since the Cytoscan measures the red blood cell column width and IFM measures luminal diameter. Thus, OPS imaging can be used to make valid measurements of microvascular diameter and red blood cell velocity in tissues. Copyright (C) 2000 S. Karger AG, Basel

Determination of regional bone blood flow by means of fluorescent microspheres using an automated sample-processing procedure

The determination of regional blood flow utilizing fluorescent microspheres (FMs) is an established method for numerous organs. Recent progress, in particular the automation of sample processing, has further improved this method. However, the FM method (reference sample technique), which allows repetitive measurement of regional organ blood flow, has so far not been used for the determination of blood flow in bone. The aim of the present study was to establish FM for the quantification of regional bone blood flow (RBBF). Female, anesthetized New Zealand rabbits (n = 6) received left ventricular injections of different amounts of FM at six subsequent time points. In order to examine the precision of RBBF determination, two different FM species were injected simultaneously at the sixth injection. At the end of the experiments the femoral and tibial condyles of each hind limb were removed and the fluorescence intensity in the tissue samples was measured by an automated procedure. In an in vitro study we have shown that acid digestion of the crystalline matrix has no effect on the fluorescence characteristics of FM. The determination of the number of spheres per tissue sample revealed that depending on the tissue sample size up to 3 x 10(6) spheres/injection were necessary to obtain about 400 microspheres in the individual bone samples. RBBF values of the tibial and femoral condyles did not differ at various injection intervals. The tibial blood flow values varied between 6.6 +/- 1.1 and 8.5 +/- 1.4 ml/min/100 g and were significantly higher than those of the femur (4.3 +/- 1.1 to 6.0 +/- 1.8 ml/min/100 g). The bone blood flow values obtained by simultaneous injection of two FM species correlated significantly (r = 0.96, slope = 1.06, intercept = 0.05), the mean difference was 0.39 +/- 1.11 ml/min/100 g. Our data demonstrate that the measurement of RBBF by means of FM allows a valid determination of RBBF. Copyright (C) 2003 S. Karger AG, Basel

Effects of NO synthase inhibitors on the synovial microcirculation in the mouse knee joint

Production of nitric oxide by the inducible NO synthase (iNOS) is known to be enhanced in chronic joint inflammation and osteoarthritis as well as aseptic loosening of joint prostheses. Initial studies yielded promising results after inhibition of the nitric oxide synthase (NOS). However, the effect of NOS inhibition has not been studied at the site of the primary function of NO, the microcirculation of the synovium in vivo. Using our recently developed model for the in vivo study of synovial microcirculation in the mouse knee joint, the effects of selective versus nonselective inhibition of iNOS were investigated by means of intravital fluorescence microscopy. After resection of the patella tendon, the synovial fatty tissue was exposed for intravital microscopy. Diameter of arterioles, functional capillary density (FCD), diameter of venules, venular red blood cell velocity and leukocyte-endothelial cell interaction were quantitatively analyzed before, and 10 and 60 min after intravenous injection of NOS inhibitors {[}selective iNOS inhibitor N-iminoethyl-L-lysine (L-NIL), and nonselective NOS inhibitor N-G-nitro-L-arginine methyl ester (L-NAME)]. Our results demonstrate that L-NAME causes a significant decrease in the arteriolar diameter and FCD associated with an increase in the leukocyte accumulation in the synovium in vivo. In contrast, L-NIL neither altered the microhemodynamics nor the leukocyte-endothelial cell interaction in the synovium, indicating its potential use for selective inhibition of iNOS in joint inflammation. Using our method, further studies will provide new insights into the unknown effect of NOS inhibition on the synovial microvasculature in inflammatory joint disease in vivo. Copyright (C) 1999 S. Karger AG, Basel

Involvement of Nitric Oxide in Microcirculatory Reactions after Ischemia-Reperfusion of the Rat Urinary Bladder

Background: Nitric oxide ( NO) plays a role in inflammation. Our aim was to investigate the role of NO in the microcirculatory changes after ischemia-reperfusion (I/R) of the bladder using intravital videomicroscopy (IVM). Methods: In rats, 60 min of bladder ischemia followed by 30 min of reperfusion was performed in the presence of N(G)-nitro-L-arginine methyl ester (L-NAME), the NO precursor L-arginine, or saline pre-treatments. Venular red blood cell velocity (RBCV), functional capillary density (FCD), vessel diameters, and leukocyte-endothelial cell interactions in postcapillary venules were determined. Concentrations of nitrite/nitrate in the plasma and myeloperoxidase (MPO) levels in the lungs and the bladder were measured. Results: Elevations of the numbers of rolling and adherent leukocytes, and of plasma nitrite/nitrate levels were found, while FCD and RBCV decreased. L-NAME pretreatment ameliorated the enhanced leukocyte-endothelial cell interactions without influencing the microcirculatory perfusion. In contrast, the L - arginine pretreatment further increased plasma nitrite/nitrate levels and preserved the FCD and RBCV, but did not affect leukocyte-endothelial interactions. None of these treatments influenced MPO activities. Conclusion: Our results suggest that NO plays an enhancing role in the I/R-induced neutrophil-endothelial interactions of the bladder. Supplementation of NO ameliorates the microcirculatory perfusion deficit without influencing the postischemic microcirculatory inflammatory reactions. Copyright (c) 2008 S. Karger AG, Base

Application of a novel method for subsequent evaluation of sinusoids and postsinusoidal venules after ischemia-reperfusion injury of rat liver

Although several intravital fluorescence microscopic studies demonstrated that microcirculatory derangement is induced during liver ischemia-reperfusion, these data were obtained from randomly selected microvascular areas and microvessels, Repeated observation of the identical microvessels has not been performed yet. Using a specially designed cover glass, it is now possible to relocate desired sites of observation repeatedly over the whole reperfusion time, The aim of this study was to determine the impact of reperfusion time on hepatic microvascular perfusion state. Twenty minutes of ischemia induced a significant decrease in sinusoidal perfusion rate (29.1 +/- 10.2%) as compared with baseline values (98.0 +/- 0.3%). At 30, 60, and 120 min of reperfusion, the percentage of perfused sinusoids recovered to 62.8 +/- 6.6, 67.5 +/- 5.7, and 77.2 +/- 5.4%. The number of stagnant leukocytes in the same sinusoids was 6.2 +/- 1.9/lobule at baseline and increased to 22.3 +/- 3.6/lobule at 120 min of reperfusion. The number of leukocytes adhering within postsinusoidal venules was 53.5 +/- 12.5/mm(2) before ischemia and increased to 414.2 +/- 62.5/mm(2) at 120 min of reperfusion. We have demonstrated that during 120 min of reperfusion, there was a steady increase in both sinusoidal and venular leukocyte adhesion along with an attenuation of the initially severely depressed sinusoidal perfusion. a no-reflow phenomenon at an early phase of reperfusion and subsequent reflow were proven

Comparison of regional blood flow values measured by radioactive and fluorescent microspheres

Fluorescent microspheres (FM) have become an attractive alternative to radioactive microspheres (RM) for the measurement of regional blood flow (RBF). The aim of the present study was to investigate the comparability of both methods by measuring RBF with FM and RM. Eight anaesthetised pigs received simultaneous, left atrial injections of FM and RM with a diameter of 15 mum at six different time points. Blood reference samples were collected from the descending aorta. RBF was determined in tissue samples of the myocardium, spleen and kidneys of all 8 animals. After radioactivity of the tissue samples was determined, the samples were processed automatically for measuring fluorescence using a recently developed filter device (SPU). RBF was calculated with both the isotope and spectrometric data of both methods for each sample resulting in a total of 10,512 blood flow values. The comparison of the RBF values yielded high linear correlation (mean r(2) = 0.95 +/- 0.03 to 0.97 +/- 0.02) and excellent agreement (bias 5.4-6.7%, precision 9.9-16.5%) of both methods. Our results indicate the validity of MS and of the automated tissue processing technique by means of the SPU. Copyright (C) 2002 S. Karger AG, Basel

Islets of Langerhans Are Protected from Inflammatory Cell Recruitment during Reperfusion of Rat Pancreas Grafts

Background: Ischemia/reperfusion (I/R) injury plays a pivotal role in the development of graft pancreatitis, with ischemia time representing one of its crucial factors. However, it is unclear, whether exocrine and endocrine tissue experience similar inflammatory responses during pancreas transplantation (PTx). This study evaluated inflammatory susceptibilities of islets of Langerhans (ILH) and exocrine tissue after different preservation periods during early reperfusion. Methods: PTx was performed in rats following 2 h (2h-I) or 18 h (18h-I) preservation. Leukocyte-endothelial cell interactions (LEI) were analyzed in venules of acinar tissue and ILH in vivo over 2 h reperfusion. Nontransplanted animals served as controls. Tissue samples were analyzed by histomorphometry. Results: In exocrine venules leukocyte rolling predominated in the 2h-I group. In the 18h-I group, additionally, high numbers of adherent leukocytes were found. Histology revealed significant edema formation and leukocyte extravasation in the 18h-I group. Notably, LEI in postcapillary venules of ILH were significantly lower. Leukocyte rolling was only moderately enhanced and few leukocytes were found adherent. Histology revealed minor leukocyte extravasation. Conclusion: Ischemia time contributes decisively to the extent of the I/R-injury in PTx. However, ILH have a significantly lower susceptibility towards I/R, even when inflammatory reactions in adjacent exocrine tissue are evident. Copyright (C) 2010 S. Karger AG, Base

Size‑specific recolonization success by coral‑dwelling damselfishes moderates resilience to habitat loss

Increasing degradation of coral reef ecosystems and specifically, loss of corals is causing significant and widespread declines in the abundance of coral reef fishes, but the proximate cause(s) of these declines are largely unknown. Here, we examine specific responses to host coral mortality for three species of coral-dwelling damselfishes (Dascyllus aruanus, D. reticulatus, and Pomacentrus moluccensis), explicitly testing whether these fishes can successfully move and recolonize nearby coral hosts. Responses of fishes to localized coral loss was studied during population irruptions of coral feeding crown-of-thorns starfish, where starfish consumed 29 (34%) out of 85 coral colonies, of which 25 (86%) were occupied by coral-dwelling damselfishes. Damselfishes were not tagged or individually recognizable, but changes in the colonization of different coral hosts was assessed by carefully assessing the number and size of fishes on every available coral colony. Most damselfishes (> 90%) vacated dead coral hosts within 5 days, and either disappeared entirely (presumed dead) or relocated to nearby coral hosts. Displaced fishes only ever colonized corals already occupied by other coraldwelling damselfishes (mostly conspecifics) and colonization success was strongly size-dependent. Despite movement of damselfishes to surviving corals, the local abundance of coral-dependent damselfishes declined in approximate accordance with the proportional loss of coral habitat. These results suggest that even if alternative coral hosts are locally abundant, there are significant biological constraints on movement of coral-dwelling damselfishes and recolonization of alternative coral habitats, such that localized persistence of habitat patches during moderate or patchy disturbances do not necessarily provide resilience against overall habitat loss

Ischemic preconditioning attenuates portal venous plasma concentrations of purines following warm liver ischemia in man

Background/Aims: Degradation of adenine nucleotides to adenosine has been suggested to play a critical role in ischemic preconditioning (IPC). Thus, we questioned in patients undergoing partial hepatectomy whether (i) IPC will increase plasma purine catabolites and whether (ii) formation of purines in response to vascular clamping (Pringle maneuver) can be attenuated by prior IPC. Methods: 75 patients were randomly assigned to three groups: group I underwent hepatectomy without vascular clamping; group II was subjected to the Pringle maneuver during resection, and group III was preconditioned (10 min ischemia and 10 min reperfusion) prior to the Pringle maneuver for resection. Central, portal venous and arterial plasma concentrations of adenosine, inosine, hypoxanthine and xanthine were determined by high-performance liquid chromatography. Results: Duration of the Pringle maneuver did not differ between patients with or without IPC. Surgery without vascular clamping had only a minor effect on plasma purine transiently increased. After the Pringle maneuver alone, purine plasma concentrations were most increased. This strong rise in plasma purines caused by the Pringle maneuver, however, was significantly attenuated by IPC. When portal venous minus arterial concentration difference was calculated for inosine or hypoxanthine, the respective differences became positive in patients subjected to the Pringle maneuver and were completely prevented by preconditioning. Conclusion: These data demonstrate that (i) IPC increases formation of adenosine, and that (ii) the unwanted degradation of adenine nucleotides to purines caused by the Pringle maneuver can be attenuated by IPC. Because IPC also induces a decrease of portal venous minus arterial purine plasma concentration differences, IPC might possibly decrease disturbances in the energy metabolism in the intestine as well. Copyright (C) 2005 S. Karger AG, Basel