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Viewing the microcirculation through the window: some twenty years experience with the hamster dorsal skinfold chamber.

Intravital microscopy represents a sophisticated technique to study the microcirculation in health and disease. While most preparations used for those studies are acute in nature, the use of chamber preparations in the skinfold bear the advantage to allow for chronic studies with repeated analysis of the microcirculation over a prolonged period of time. The skinfold chamber model for microcirculatory analysis has been adapted to mice, rats and hamsters. Although the use of rats and, in particular, the use of mice has the advantage of the availability of species-specific tools, the use of the hamster as the experimental animal may be preferred due to anatomical reasons, which facilitate the microsurgical preparation and improve the quality of microscopic imaging. The use of the hamster dorsal skinfold chamber, firstly described by Endrich and coworkers in 1980, has brought out during the last two decades a considerable number of experimental studies within the fields of microcirculation physiology, inflammation and sepsis, ischemia-reperfusion, angiogenesis, and transplantation, indicating that the model has to be considered a versatile tool to study the microcirculation in health and disease.

Animals↗

Comparison of the new OPS imaging technique with intravital microscopy: analysis of the colon microcirculation.

BACKGROUND: The OPS imaging technique has been introduced for in vivo assessment of microcirculation in humans. The aim of this study was to validate the new technique against intravital fluorescence microscopy (IFM) for the visualization of colon microcirculation in a murine model of inflammatory bowel disease (IBD). METHOD: IBD was induced in Balb/c mice by dextran sulfate sodium, controls received normal water. In each animal, both the CYTOSCAN A/R and IFM were used to image the microcirculation (n = 7 in each group). The postcapillary venular diameter was analyzed on the colon muscularis and mucosa. RESULTS: The venular diameter correlated significantly between both methods representing the good correspondence between both methods. CONCLUSION: Our study demonstrates that the new technique for visualization of microcirculation without use of fluorescent dyes, the OPS imaging, allows for quantitative measurement of a key microcirculatory parameters of the mouse colon.

Animals↗

Direct in vivo visualization of renal microcirculation by intravital CCD videomicroscopy.

Several types of experimental techniques have been developed for the evaluation of renal microcirculation. Although each methodology possesses excellent and unique characteristics, it requires substantial artificial manipulation that might alter the renal microvascular responsiveness. To circumvent such limitation of previous ex vivo or in vitro approaches to glomerular microcirculation, we have developed a pencil lens-probe charge-coupled device (CCD) intravital videomicroscopic system which allows us to evaluate both systemic hemodynamics and renal microcirculation. Furthermore, real-time images of afferent and efferent arterioles as well as glomeruli can be continuously assessed, which would facilitate the functional characterization of these microvessels in vivo. Finally, the tapered nature of the CCD probe of this videomicroscopy may allow direct observation of the renal microvasculature in small animals (e.g., rats and mice). In conclusion, this novel technique is a valuable tool for unveiling the in vivo, in situ and intact renal microvascular behavior, and may provide further approaches to the understanding of renal microcirculation.

Animals↗

Platelet kinetics in the pulmonary microcirculation in vivo assessed by intravital microscopy.

Growing evidence supports the substantial pathophysiological impact of platelets on the development of acute lung injury. Methods for studying these cellular mechanisms in vivo are not present yet. The aim of this study was to develop a model enabling the quantitative analysis of platelet kinetics and platelet-endothelium interaction within consecutive segments of the pulmonary microcirculation in vivo. New Zealand White rabbits were anesthetized and ventilated. Autologous platelets were separated from blood and labeled ex vivo with rhodamine 6G. After implantation of a thoracic window, microhemodynamics and kinetics of platelets were investigated by intravital microscopy. Velocities of red blood cells (RBCs) and platelets were measured in arterioles, capillaries and venules, and the number of platelets adhering to the microvascular endothelium was counted. Kinetics of unstimulated platelets was compared with kinetics of thrombin-activated platelets. Velocity of unstimulated platelets was comparable to RBC velocity in all vessel segments. Unstimulated platelets passed the pulmonary microcirculation without substantial platelet-endothelial interaction. In contrast, velocity of activated platelets was decreased in all vascular segments indicating platelet margination and temporal platelet-endothelium interaction. Thrombin-activated platelets adhered to arteriolar endothelium; in capillaries and venules adherence of platelets was increased 8-fold and 13-fold, respectively. In conclusion, using intravital microscopy platelet kinetics were directly analyzed in the pulmonary microcirculation in vivo for the first time. In contrast to leukocytes, no substantial platelet-endothelium interaction occurs in the pulmonary microcirculation without any further stimulus. In response to platelet activation, molecular mechanisms enable adhesion of platelets in arterioles and venules as well as retention of platelets within capillaries.

Animals↗

OPS imaging of human microcirculation: a short technical report.

Despite the pivotal role of microcirculation in numerous diseases, techniques for the direct assessment of human microcirculation are limited. A new approach based on orthogonal polarization spectral (OPS) imaging (Cytoscan microscope) allows noninvasive observation of human microcirculation in all accessible tissue surfaces. Limitations remain: application of pressure with the instrument affects blood flow, lateral movement of tissue precludes continuous investigation of a given microvascular region, and blood flow velocities above 1 mm/s cannot be measured. We addressed these problems by (a) constructing an attachment to the probe, preventing direct contact of the instrument with the observed tissue area and allowing fixation of the tissue, and (b) implementing a double-flash spatial correlation technique extending the measuring range for blood flow velocities up to approximately 40 mm/s. The modified approach was tested in vitro and in vivo. Velocity readings correlated well with velocities of an external standard (r(2) = 0.99, range 1.9-33.8 mm/s). Pulsatile flow patterns synchronous with heart rate with maximal velocities of about 10 mm/s could be detected in arterioles of the human sublingual mucosa. The modified instrument may prove useful to investigate the microcirculation in the context of research, diagnosis and therapy control.

Adult↗

Improvement of impaired microcirculation and tissue oxygenation by hemodilution with hydroxyethyl starch plus cell-free hemoglobin in acute porcine pancreatitis.

AIMS: To avoid the progression from mild edematous acute pancreatitis (AP) to the severe necrotizing form, one therapeutic option is to improve pancreatic microcirculation and tissue oxygenation. The aim of the study was to evaluate the influence of improved rheology (isovolemic hemodilution) plus enhanced oxygen supply (bovine hemoglobin HBOC-301) on pancreatic microcirculation, tissue oxygenation and survival in severe acute experimental pancreatitis. METHODS: Severe AP was induced in 39 pigs (25-30 kg BW) by stimulation with intravenous administration of cerulein plus a pressure- and volume-controlled 10-min intraductal infusion of glycodeoxycholic acid. Seventy-five minutes after induction of AP, animals were randomized and hemodiluted isovolemically (PAOP constant) with either 10% hydroxyethyl starch (HES) 200,000/0.5 plus HBOC-301 (+0.6 g/dl plasmatic hemoglobin; Oxyglobin, Biopure, Cambridge, Mass., USA), or 10% HES 200,000/0.5, or Ringer's solution to a hematocrit of 15%. Hemodynamics, oxygen transport parameters, pancreatic microcirculation and tissue oxygen tension were evaluated over 6 h. Then the abdomen was closed, animals were extubated and observed for 6 days. After that, the surviving animals were sacrificed and specimens were taken from the pancreas. The histopathologic findings were scored by two blinded pathologists who quantified acinar necrosis, fat necrosis, inflammation and edema. RESULTS: Isovolemic hemodilution with HES plus HBOC-301 reduced mortality and preserved pancreatic microcirculation compared with Ringer's solution, but was not significantly different from hemodilution with HES alone. Only treatment with HES plus HBOC-301 normalized pancreatic tissue oxygen tension compared with IHD with HES or Ringer's solution alone. CONCLUSIONS: IHD with HES plus HBOC-301 as a combination of rheologic and O(2)-delivering therapy may represent a novel therapeutic option for treatment of AP.

Acute Disease↗

Methodological approaches used for the study of the coronary microcirculation in situ.

Measurements of coronary microvascular parameters in situ are difficult because of the thickness of the heart muscle and cardiac contraction. Both of these problems hamper the visualization of the coronary microcirculation. We have refined methodological approaches that enable the study of the coronary microcirculation in situ. In the first approach, microvessels can be visualized in the beating heart using a preparation that compensates for cardiac motion by creating an illusion that the heart is motionless. This is accomplished by flashing a stroboscopic light source once per heart cycle at the same point in each cycle and synchronizing a ventilator with the cardiac cycle. Images of microvessels can be obtained using standard intravital video-microscopic techniques. To visualize the intramural and subendocardial microcirculation, studies are completed in isolated hearts. In this preparation, measurements of microvascular diameters and pressures can be performed in both the subepicardial and subendocardial microcirculations. This latter approach allows insight into transmural differences of coronary microvascular regulation.

Animals↗

Influence of fluid removal during haemodialysis on macro- and skin microcirculation. Haemodynamic pathophysiologic study of fluid removal during haemodialysis.

Changes of macro- and microcirculation during haemodialysis and fluid removal are probably dependent on ultimate fluid status and on the efficacy of various regulation mechanisms, especially the catecholamines. This was studied in 20 chronic dialysis patients. Pre- and postdialysis stroke volume, mean arterial pressure, heart rate and systemic vascular resistance were measured. Furthermore, microcirculation was studied by Laser Doppler flow and by intravital microscopy of finger nail fold, measuring red blood cell velocity and capillary density. Pre- and postdialysis noradrenaline and adrenaline were measured. Nine patients proved to be hypovolaemic after dialysis (group I) and 11 patients proved to be normovolaemic or less hypervolaemic (group II) according to vena cava inferior parameters. There was a significant decrease of mean arterial pressure and stroke volume in group I, and an increase of heart rate, whereas in group II there was only a decrease of mean arterial pressure. Systemic vascular resistance did not change in both groups. Noradrenaline decreased although not significantly in both groups, whereas in group I adrenaline increased significantly. There was a significant decrease of skin perfusion in group I, whereas in group II there was a significant increase. Capillary density increased significantly in group II after reaching normovolaemia. Underhydration was leading to a decrease of skin microcirculation on the basis of a decrease of stroke volume and an increase of adrenaline levels. In hypervolaemic patients, who were ultrafiltrated to normovolaemia, skin microcirculation improved on the basis of a decrease of arterial and venous pressure and consequently a decrease of the myogenic response as a local autoregulatory effect.

Adult↗

Novel index for invasively assessing the coronary microcirculation.

BACKGROUND: A relatively simple, invasive method for quantitatively assessing the status of the coronary microcirculation independent of the epicardial artery is lacking. METHODS AND RESULTS: By using a coronary pressure wire and modified software, it is possible to calculate the mean transit time of room-temperature saline injected down a coronary artery. The inverse of the hyperemic mean transit time has been shown to correlate with absolute flow. We hypothesize that distal coronary pressure divided by the inverse of the hyperemic mean transit time provides an index of microcirculatory resistance (IMR) that will correlate with true microcirculatory resistance (TMR), defined as the distal left anterior descending (LAD) pressure divided by hyperemic flow, measured with an external ultrasonic flow probe. A total of 61 measurements were made in 9 Yorkshire swine at baseline and after disruption of the coronary microcirculation, both with and without an epicardial LAD stenosis. The mean IMR (16.9+/-6.5 U to 25.9+/-14.4 U, P=0.002) and TMR (0.51+/-0.14 to 0.79+/-0.32 mm Hg x mL(-1) x min(-1), P=0.0001), as well as the % change in IMR (147+/-66%) and TMR (159+/-105%, P=NS versus IMR % change), increased significantly and to a similar degree after disruption of the microcirculation. These changes were independent of the status of the epicardial artery. There was a significant correlation between mean IMR and TMR values, as well as between the % change in IMR and % change in TMR. CONCLUSIONS: Measuring IMR may provide a simple, quantitative, invasive assessment of the coronary microcirculation.

Animals↗

Hemodynamic characteristics of the intestinal microcirculation in renal hypertension.

This study investigated the microvascular changes that affect vascular resistance in the rat small intestine during two-kidney, one clip renal hypertension 4 weeks after renal artery stenosis. To study the intestinal microcirculation, a loop of the small intestine was exteriorized with intact circulation and innervation and a section of the bowel wall was prepared for observation with an intravital video microscopy system. Microvascular diameter, pressure, and flow velocity were measured for first, second, and third branch order arterioles and venules, using an image shearing monitor, servo-null micropipette system, and an optical Doppler velocimeter, respectively. The diameters of the first order arterioles and venules were significantly (p less than 0.05) reduced in hypertensive rats; however, diameters were unaltered in smaller second and third order arterioles and venules as compared with normotensive vessels. In hypertensive rats, mean arterial pressure was significantly (p less than 0.05) elevated (47%) and pressures also were elevated significantly (p less than 0.05) throughout the microcirculation, although by a proportionally smaller amount. Total network flow (i.e., first order arteriole flow) was significantly (p less than 0.05) reduced (40%) in hypertensive rats, but volume flows in individual second and third order arterioles were similar to flows measured in normotensive rats. Calculated total network resistance was increased (124%) in hypertensive rats. Thus, the intestinal microcirculation in rats with two-kidney, one clip renal hypertension is disturbed by elevated pressure and decreased total flow. The presence of normal flows in individual second and third order arterioles without any demonstrable difference in their diameters suggests that the predominant cause of elevated resistance across this segment of the intestinal microcirculation is a reduction in the number of perfused small arterioles.

Animals↗

Microcirculation as a novel marker of membrane biocompatibility.

BACKGROUND: It is possible to consider microcirculation as a kind of OwitnessO of the complex biological reactions triggered by the dialytic treatment. The reactivity of microcirculation to the dialytic stress may represent a measure of the overall biocompatibility of the membrane. In this study we tested the hypothesis that different synthetic membranes may have different biological effects, particularly related to microcirculation. SUBJECTS AND METHODS: In this crossover study, we observed 16 chronically hemodialyzed patients. All patients were treated with the EVAL membrane; we recorded the TcPO2 during the second treatment of the week. All patients were then switched to the hf-PS membrane. During the study observation we did not change the dialytic prescription or the pharmacologic treatment. RESULTS: From the beginning of the session until 90O, the behavior of TcPO2 is similar for both the membranes. From 120O to the end of the treatment in sessions with the EVAL membrane, the TcPO2 values come back to the starting level, whereas in the treatments with hf-PS the TcPO2, the values remain at a lower level; there was a significant difference between EVAL and hf-PS in the values recorded. Arterial blood gas values of paO2 and paCO2 are quite similar in the treatments with both the membranes, without any significant difference. CONCLUSIONS: The analysis of microcirculation by means of TcPO2 measurement is a useful tool to obtain a OclinicalO measure of biocompatibility of the dialytic treatment and different membranes may have different impacts on TcPO2.

Aged↗

Activated protein C improves intestinal microcirculation in experimental endotoxaemia in the rat.

INTRODUCTION: Successful treatment of severe sepsis and septic shock remains a major challenge in critical care medicine. The recently introduced recombinant human activated protein C (APC) remarkably improved the outcome of septic patients. The influence of APC on intestinal circulation is still poorly understood. Therefore, the present study aimed to investigate the effects of APC on intestinal microcirculation during experimental endotoxaemia in rats by using intravital microscopy. METHODS: A total of 44 male Lewis rats were randomly assigned to receive intravenous injections of 15 mg/kg lipopolysaccharide alone (LPS) (n = 11) or LPS followed by subsequent injection of 2 mg/kg recombinant human APC (LPS + APC) (n = 11), whereas control animals received either APC (n = 11) or saline (n = 11). Animals underwent observations of functional capillary density and leucocyte adherence on venular endothelium in the microcirculation of the intestinal wall by means of intravital fluorescence microscopy. Indicators of macrocirculation as well as plasma levels of tumour necrosis factor-alpha, interleukin (IL)-1beta, IL-6, and IL-10 were measured. RESULTS: Although APC administration of both LPS-treated and control rats did not change macrocirculation or release of inflammatory cytokines, it increased mucosal and muscular functional capillary density (p < 0.001 and p < 0.05, respectively) and reduced the number of firmly adhering leucocytes in intestinal submucosal V1 and V3 venules (p < 0.01) in LPS + APC-treated compared with LPS-treated animals, which did not receive APC. No remarkable differences that could be attributed to APC treatment were observed between the two control groups. CONCLUSION: APC administration during experimental endotoxaemia improved intestinal microcirculation by protecting functional capillary density as a measure of microvascular perfusion and exerted anti-inflammatory effects by reducing leucocyte adherence to the endothelium in submucosal venules. Therefore, beneficial effects of APC in septic patients might be due, in part, to improved intestinal microcirculation.

Animals↗

Arteriolovenular shunting critically determines shutdown of microcirculation upon cryotherapy in tumor-bearing rat liver.

BACKGROUND: Tissue destruction by cryosurgery not only is mediated by direct cell damage, but also involves secondary mechanisms, such as ischemia due to shutdown of the microcirculation. Clinicians favor repetitive cryoapplication, although there is no proven evidence for a more effective tumor eradication. METHODS: The aims of this study were (1) to establish a rat liver tumor model that allows for intravital microscopic analysis of hepatic tumor microcirculation and (2) to elucidate critical determinants of shutdown of microvascular perfusion after single and repetitive cryotherapy. In WAG-Rji rats (n = 14), syngeneic colon carcinoma cells (CC531) were implanted into the left liver lobe. Hepatic and tumor microcirculation were studied by intravital microscopy. RESULTS: Two weeks after implantation, the tumors had developed a microvasculature with a capillary density markedly (P < .05) lower compared with the sinusoidal density of normal liver. However, at the tumor margin, venule diameters were significantly enlarged (P < .05), with high red blood cell velocities and arteriolovenular shunts. Both freeze procedures (temperature at the tumor margin: -32.4 degrees C +/- 1.6 degrees C and -36.4 degrees C +/- 2.0 degrees C) resulted in a complete shutdown of intratumoral and peritumoral capillary and hepatic sinusoidal perfusion. In contrast, some large venules showed maintenance of blood flow initially after freezing (15 minutes); however, this was abolished during the subsequent 2-hour observation period. CONCLUSIONS: Enlarged high-flow venules at the tumor margin, which participate in arteriolovenular shunting, critically determine the shutdown of the microcirculation upon cryotherapy. Repetitive freezing is not more effective than a single-freeze procedure to achieve complete tumor microcirculatory stasis.

Animals↗

[Renal microcirculation].

The blood flow to the kidney averages about 20% of the cardiac output and the renal circulation affects urine formation. The renal microcirculation is unique. Glomerular circulation is mainly regulated by two resistance arterioles, the afferent arteriole and the efferent arteriole. Two capillary beds are arranged in series between the arterial and venous circulation. Many experimental findings show that each arteriole has a different sensitivity to various stimuli. Moreover, several anatomic characteristics of the renal circulation display heterogeneity. Several regional differences in the renal microcirculation may subserve the excretory function of the kidney. Technological advances now permit direct observation of renal arterioles and measurements of microvascular pressure, flows and resistances. In this mini review, we introduce 1) characteristics of renal circulation, 2) techniques for study of renal microcirculation, 3) effects of angiotensin II on the renal microvasculature, and 4) future directions for the study of renal microcirculation.

Angiotensin II↗

Analysis of the derangement of the pancreatic microcirculation in a rat caerulein pancreatitis model using an intravital microscope system.

In order to clarify the derangement of the pancreatic microcirculation in acute pancreatitis, the pancreatic microcirculation in caerulein pancreatitis was monitored intravitally and the roles of bradykinin and nitric oxide were examined using bradkykinin B2 receptor antagonist, HOE140. Under an intravital microscope, the pancreatic microcirculation was observed 2 or 6 hr after the induction of acute pancreatitis. HOE140 was administered 30 min before the induction of acute pancreatitis. The videoimages were taken into the computer, and the value of grayscale was measured with imaging software to quantify the degree of extravasation. Extravasation in the postcapillary venules was remarkable and the velocity in pancreatic terminal arterioles decreased significantly. However, the adherence of leukocytes was not observed until 6 hr after the induction. Both the extension of extravasation and the decrease of velocity were prevented by HOE140. The levels of nitric oxides in the pancreatic tissue declined and this decline was not influenced by HOE140. Bradykinin participates mainly in the regulation of vascular permeability in the early stage of caerulein pancreatitis. Further, the impairment of pancreatic microcirculation may play a key role in the onset and development of acute pancreatitis.

Acute Disease↗

Renal microcirculation in experimental acute pancreatitis of dogs.

In order to understand the mechanism of acute renal failure frequently observed in severe acute pancreatitis, renal microcirculation and renal hemodynamics were investigated during experimental acute pancreatitis in dogs induced by autologous bile and trypsin mixture into the pancreatic duct. Renal tissue blood flow (hydrogen gas clearance method), renal arterial blood flow, and cardiac output (transonic blood flow meter) were each measured for 5 h after induction of pancreatitis. The effect on renal hemodynamics of a new synthesized protease inhibitor--E-3123; 4-(2-succinimidoethylthio)phenyl-4-quanidinobenzoate methane sulfonate--intravenously infused at the rate of 3 mg/kg/h was also investigated. The mean blood pressure and pulse pressure decreased after induction of pancreatitis. Renal microcirculation and renal artery blood flow decreased during the experiment. However, in dogs with treated by E-3123, renal microcirculation was preserved during the first hour of the experiment and decreased gradually afterward, but it was significantly higher than that of the dogs without E-3123 during 3-5 h. The mean blood pressure and pulse pressure were preserved nearly at preoperative levels during the experimental period. We concluded that renal microcirculation decreased concomitantly with a deterioration of acute pancreatitis, and that the new pancreatic protease inhibitor E-3123 may have some beneficial effect to improve renal hemodynamics in the early period of acute pancreatitis.

Acute Disease↗

Differences in platelet endothelial cell adhesion molecule-1 expression between peripheral circulation and pancreatic microcirculation in cerulein-induced acute edematous pancreatitis.

AIM: To investigate the changes of platelet endothelial cell adhesion molecule-1 (PECAM-1) expression on polymorphonuclear leukocytes (PMNs) in peripheral circulation and pancreatic microcirculation in cerulein-induced acute edematous pancreatitis (AEP). METHODS: Fifty Wistar rats were randomly divided into control group (n = 10) and AEP group (n = 40). A model of AEP was established by subcutaneous injection of cerulein 5.5 and 7.5 mug/kg at 0 and 1 h after the beginning of experiment respectively. PECAM-1 expression on PMNs from splenic vein and inferior vena cava was determined by RT-PCR at mRNA level and determined by flow cytometry at protein level. RESULTS: In experimental rats, an increased PECAM-1 mRNA expression was seen from 4 to 8 h of AEP in peripheral circulation (0.77+/-0.25%, 0.76+/-0.28%, 0.89+/-0.30%, 1.00+/-0.21%), while in pancreatic microcirculation, expression decreased from 2 h and reached the lowest level at 6 h of AEP (0.78+/-0.29%, 0.75+/-0.26%, 0.62+/-0.28%, 0.66+/-0.20%). There were significant differences at 8-h time point of AEP between peripheral circulation and pancreatic microcirculation (1.00+/-0.21% vs 0.66+/-0.20%, P<0.05). Meanwhile, the difference at protein level was also found. CONCLUSION: A reverse expression of PECAM-1 on PMNs was found between peripheral circulation and pancreatic microcirculation, suggesting that inhibition of PECAM-1 expression may improve the pathological change of AEP.

Acute Disease↗

[Coronary microcirculation. Physiologic and pathologic aspects].

The coronary circulation has a protective regulation system which, in extreme haemodynamic conditions, compensates increased myocardial oxygen demand. The coronary reserve, based on this concept defines the capacity of the system to increase flow temporally, and, thereby, myocardial oxygen supply. The introduction of new methods of investigating the coronary microcirculation has enabled the study of this phenomenon in several cardiovascular pathologies. Two types of investigation are used currently for studying the coronary microcirculation: 1) invasive methods, especially the recently developed intracoronary Doppler and pressure guide, 2) non-invasive methods, and, in particular, contrast echocardiography, position emission tomography and magnetic nuclear resonance. These investigations allow measurement of the coronary reserve or the assessment of the myocardial consequences of abnormalities of the microcirculation. Some workers use these methods to investigate pathological coronary microcirculation in different cardiomyopathies, in the presence of different cardiovascular risk factors (hypertension, diabetes, smoking, hypercholesterolaemia) and after cardiac transplantation.

Coronary Circulation↗