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PubMed2003

Prototype of an in vitro model of the microcirculation.

We have used microfabrication technology to construct a network of microchannels, patterned after the dimensions and architecture of the mammalian microcirculation. The network is cast in transparent silicone elastomer and the channels are coated with silanated mPEG to provide lubrication. Flow of red and white blood cells through the network is readily visualized by the use of high-speed digital image acquisition. The acquired sequences of high-quality images are used to calculate hematocrits and rates of red cell movement in the microchannels. Our prototype system has significant advantages over scaled-up room-size experimental systems in that it permits experimentation with actual human blood cells. Experiments can be carried out under well-controlled conditions in a network of microchannels with precisely known dimensions using cell suspensions of defined composition. Moreover, there is no need to counteract or anticipate the host's adaptive responses that may confound live animal experiments. Notwithstanding its limitations, the current prototype demonstrates certain features characteristic of the microcirculation, such as parachute and bullet shapes of red cells deformed in capillary channels, rouleaux formation, plasma skimming, and the utilization of collateral flow pathways due to flow obstruction caused by a white cell blocking a microchannel. We present this device as a prototype scale-to-scale model of the mammalian microcirculation. Limitations of the system as well as a variety of possible applications are described.

Animals↗

PubMed2001

Microcirculation patterns other than loops and networks in choroidal and ciliary body melanomas.

PURPOSE: To provide ophthalmologists and pathologists with expanded criteria for separating patients at high risk of metastatic melanoma from those at low risk on the basis of microcirculation patterns in choroidal and ciliary body melanomas. DESIGN: Tissue culture studies and observational case series. PARTICIPANTS: The pattern-forming ability of four uveal melanoma cell lines of varying degrees of aggressive behavior was studied in vitro. Histologic sections of 234 eyes removed for choroidal or ciliary body melanoma were studied for the presence of microcirculation patterns. METHODS: The study was divided into two phases: the study of histologic sections of eyes removed for choroidal and ciliary body melanomas and observations on the in vitro behavior of cultured melanoma cells of varying degrees of invasive behavior. The presence or absence of each of nine microcirculation patterns was recorded from tissue sections, and interrelationships between different patterns were explored statistically. In vitro reconstitution of patterns and a study of the interrelationships of patterns in histologic sections was carried out. In the in vitro studies, uveal melanoma cell lines of varying degrees of aggressive potential were cultured to observe the development of architectural patterns other than loops and networks. MAIN OUTCOME MEASURES: In histologic studies, the outcome measure was the conditional probability of detecting loops or networks given the presence or absence of other patterns positive for periodic acid-SCHIFF: For tissue culture studies, the outcome measure was either the development or lack of development of patterns of different shapes in vitro. RESULTS: Histologic studies disclosed that given the presence of arcs without or with branching in a tissue section, it is likely that loops or networks will be detected in the same section plane, suggesting that the production of these patterns by aggressive tumor cells reflects a spectrum of architectural potential. In vitro studies confirmed this hypothesis by revealing that highly aggressive and metastatic uveal melanoma cell lines, but not poorly aggressive tumor cell lines, generated parallel channels with and without crosslinking and arcs with and without branching as well as loops and networks. CONCLUSIONS: The criteria for separating patients into low- and high-risk categories for metastasis from uveal melanoma should be expanded to include patterns other than loops or networks. In both the pathology laboratory as well as in a clinical setting, the detection of arcs or arcs with branching and parallel channels should prompt a careful search for loops and networks and for crosslinking parallel channels, respectively.

Choroid Neoplasms↗

PubMed1997

Nicotine induces leukocyte rolling and adhesion in the cerebral microcirculation of the mouse.

Nicotine and several related metabolites were examined for their ability to induce leukocyte rolling and adhesion in the cerebral microcirculation of the mouse. A cranial window was surgically prepared for the visualization of the pial microcirculation using an intra-vital microscopy imaging system. Using this technique rhodamine-labeled leukocytes could be visualized and video-recorded as they traveled within the microvessels, and the quantitation of their rolling and adhesion along the pial venule walls was assessed during an off-line video playback analysis. Nicotine was found to produce significant dose-related increases in leukocyte rolling and adhesion. Cotinine, a major nicotine metabolite, did not induce the same degree of leukocyte rolling and adhesion. Mecamylamine, a nicotine antagonist, was found to inhibit the nicotine-induced leukocyte rolling and adhesion. Anti-P-selectin antibody blocked nicotine-induced leukocyte rolling, while anti-CD18 antibody effectively inhibited leukocyte adhesion, but not rolling in similar experiments. Nicotine-induced leukocyte rolling and adhesion were also inhibited by superoxide dismutase and catalase. These data suggest that nicotine, the principle pharmacological agent in cigarette smoke and related tobacco products, acts via a ganglionic-type nicotinic receptor to enhance leukocyte rolling via P-selectin and reactive oxygen radical-dependent mechanisms in cerebral microcirculation of the mouse.

Administration, Topical↗

PubMed2000

High-frequency color flow imaging of the microcirculation.

The extension of ultrasound (US) color flow imaging (CFI) techniques to high frequencies (> 20 MHz) has the potential to provide valuable noninvasive tools for scientific and clinical investigations of blood flow in the microcirculation. We describe the development of a slow-scan CFI system operating in the 20-100-MHz range that has been optimized to image the microcirculation. The apparatus has incorporated elements of a previously reported pulsed-wave Doppler system and is capable of operating in either CFI or pulsed-wave mode. The performance of the CFI system was evaluated at a center frequency of 50 MHz using two PVDF transducers with -6-dB beam widths of 43 and 60 microm. The -6 dB-axial resolutions were estimated to be 66 and 72 microm, respectively. In vivo validation experiments conducted using the murine ear model demonstrated the detection of flow in vessels down to 15-20 microm in diameter with flow velocities on the order of mm per s. Further experiments examining experimental murine tumors confirmed the successful detection of flow in the tumor microcirculation.

Animals↗

PubMed1997

Intracranial meningeomas: time- and dose-dependent effects of irradiation on tumor microcirculation monitored by dynamic MR imaging.

The purpose of this study was the characterization of the time- and dose-dependent effects of irradiation on tumor microcirculation by means of dynamic MR imaging and correlation of the estimated data with tumor response in patients with meningeomas. Dynamic MR imaging studies were performed in 20 patients with intracranial meningeomas prior to (n = 20) and at 6 (n = 17), 18 (n = 17), and 50 wk (n = 14) after the end of radiotherapy. In seven of these patients, dynamic measurements were also performed during fractionated radiotherapy after approximate 20 Gy and 54 Gy. During and after short-time infusion of gadopentetate dimeglumine, the kinetics of lesion response was resolved using a strongly T1-weighted saturation recovery TurboFLASH (SRTF) sequence. The signal-time courses of the suspected lesions were analyzed using a pharmacokinetic two-compartment model. The calculated parameters amplitude A (reflecting gadopentetate dimeglumine accumulation in the extracellular space) and exchange rate constant k21 (depending on vascular permeability and blood flow) were displayed as color-coded images and analyzed as a function of time of therapy and radiation dose. All meningeomas showed a high exchange rate constant k21 (median, 5.7 min-1; range, 1.9-23.0 min-1) and a high amplitude A (median, 1.5 arbitrary units; range, 1.1-2.7) prior to X-ray treatment. During radiotherapy we found a dose related significant (p < .01) increase of k21 accompanied by an increase of the amplitude A as compared to the pretreatment values. Analysis of tumor volume 6, 18, and 50 wk after X-ray treatment revealed two different groups. In the responder group (n = 13) the median of the tumor volume decreased from 10.0 to 7.5 cm3. For this group, we found a significant drop (p < .01) of the median of the amplitude A and a decrease of the exchange rate constant k21. In the nonresponder group (n = 4) the median of the tumor volume increased after radiation from 3.5 to 4.5 cm3. The pharmacokinetic analysis revealed a decrease of the amplitude A-and an increase of the exchange rate constant k21. The response of meningeomas to radiotherapy is influenced by the effect of X-rays on tumor microcirculation. This effect on tumor microcirculation can be derived by analysis of pharmacokinetic maps obtained from dynamic MR images. Furthermore, these pharmacokinetic maps can possibly be used to differentiate groups of patients who respond or do not respond to radiotherapy and, thus, could benefit from another treatment modality.

Contrast Media↗

PubMed1998

Endothelium-dependent vasodilatation is impaired in both microcirculation and macrocirculation during acute hyperglycemia.

PURPOSE: Endothelial dysfunction is associated with atheromatosis and is a common finding with diabetes. We have studied the effects of acute hyperglycemia on the endothelium-dependent vasodilatation of both the microcirculation and the macrocirculation of healthy subjects. Because of the presence of endothelial dysfunction with diabetes, we hypothesize that acute hyperglycemia causes impaired endothelial-dependent responses. METHODS: Twenty healthy subjects (15 men, 5 women) with a mean age of 32.3 years (range, 23 to 49 years) were examined during fasting conditions and at 1 hour after the ingestion of 75 g of glucose. The endothelium-dependent vasodilatation of the brachial artery, a conduit vessel, was evaluated with high-resolution ultrasound scan to measure the changes in the vessel diameter induced with reactive hyperemia. In the microcirculation, the endothelial function was assessed by measuring the changes in the erythrocyte flux after the acetylcholine iontophoresis. RESULTS: The brachial artery endothelium-dependent dilatation was greater during fasting as compared with the response after the glucose load was administered (11.7% [8.3 to 14.3] vs 4.2% [1.5 to 9.6]; P < .001; median, first, and third quartile). Both peak and average blood flow velocities during the hyperemic response were higher after the administration of the glucose load as compared with the fasting period (P < .05), but no changes were found in the blood flow volume. During fasting, microcirculatory endothelial-dependent vasodilatation was also significantly greater than the response after the administration of the glucose load (1293% [591 to 1856] vs 863% [385 to 1180]; P < .01). CONCLUSIONS: In healthy subjects, the ingestion of a glucose load impairs the endothelial-dependent vasodilation in both the microcirculation and the macrocirculation. Because impairment of endothelial responses is associated with the early changes of atherosclerosis, it is possible that prolonged hyperglycemia and endothelial dysfunction may lead to the early and accelerated atherosclerosis of diabetes. Further studies are necessary to examine the long-term effects of hyperglycemia.

Acute Disease↗

PubMed1995

Cocaine and the porcine coronary microcirculation: effects of chronic cocaine exposure and hypercholesterolemia.

OBJECTIVE: To examine the acute effect of cocaine on the coronary microcirculation and whether chronic cocaine administration with or without a concomitant high-cholesterol diet affects beta-adrenoceptor and endothelial functions in the coronary microcirculation. DESIGN: Prospective experimental study. SETTING: Laboratory and animal research facility. PARTICIPANTS: Yorkshire pigs. INTERVENTIONS: Pigs were fed a high (2%)-cholesterol diet or a regular diet for 3 months. Animals in both groups received cocaine chronically (7 mg/kg/day, IM). Control animals were fed a regular diet. MEASUREMENTS AND MAIN RESULTS: Responses of the porcine coronary arterioles (90 to 190 microns in diameter) were examined in vitro in a pressurized (40 mmHg) no-flow state using a video-imaging apparatus. Acute application of cocaine caused a significant contraction with a mean maximal diameter decrease of 14% +/- 5%, which was markedly reduced by muscarinic blockade but not significantly affected by alpha 1-adrenergic blockade. Lidocaine or procainamide had no vasoconstrictor effect. Chronic exposure of animals to cocaine diminished contractile responses to cocaine and reduced relaxation responses to the nonselective beta-adrenergic receptor agonist isoproterenol. Phenylephrine caused a minimal (< 4%) contraction of vessels in all groups. Chronic cocaine administration with concomitant high-cholesterol feeding attenuated endothelium-dependent relaxations to serotonin, whereas endothelium-dependent relaxations to bradykinin were unaffected. Endothelium-independent relaxations to sodium nitroprusside were similar in all groups. CONCLUSIONS: These results suggest that cocaine can exert a direct vasoconstrictor effect on the porcine coronary microcirculation via a muscarinic mechanism. Chronic exposure to cocaine significantly decreases beta-adrenoceptor-mediated relaxation and blunts endothelium-dependent relaxation to a small degree.

Adrenergic alpha-Antagonists↗

PubMed1995

Effects of compression and type of bed surface on the microcirculation of the heel.

OBJECTIVE: To assess the effects of compression on the skin microcirculation of the heel using laser Doppler fluxmetry. DESIGN: Parallel groups comparing patients with control groups. SETTING: Department of Surgery, University College London Medical School, London. SUBJECTS AND MATERIALS: Ten patients at risk of developing pressure ulceration, 10 age- and sex-matched healthy subjects and 10 young, healthy volunteers. An acrylic indenter with a slot to accommodate a laser Doppler probe was used to apply compression to the heel region. A pressure sensor was used to measure the applied compression. OUTCOME MEASURES: The resting laser Doppler flux was measured with the subject lying supine. Compression forces were then applied in increments from 50 g to 1500 g and the corresponding interface pressure (IP) and laser Doppler flux (LDF) recorded. The IP and LDF were also measured from the heel while the subject was lying on a low air-loss system and then on an NHS conventional hospital bed. RESULTS: The resting LDF is lower in the patient group compared to the control groups (p < 0.05). Compression of the heel caused a progressive decrease in LDF in all groups. Compression greater than 50 mmHg as well as lying on an NHS bed reduced the LDF signal to a minimal value (biological zero). On the low air-loss system, the median LDF was 17% of the resting value in the age-matched control group and 32% in the patient group. CONCLUSIONS: The results indicate that the heel microcirculation is vulnerable to compression. The low air-loss system maintained the IP sufficiently low to prevent complete cessation of the heel microcirculation.

Adult↗

PubMed2000

Autonomic nervous function assessment using thermal reactivity of microcirculation.

There are only a few reliable objective methods of diagnosing peripheral neuronal damage suitable for routine use; the most important is based on measurement of nerve conduction velocity, which only shows changes when severe disturbances are already present. However, it is precisely at this stage that the possibilities of therapy are no longer satisfactory. As small fibres are affected earlier in the course of most forms of PNP than the large ones, assessment of afferent as well as efferent C-fibre function gains importance in the management of this widespread disease. In assessment of autonomic dysfunction, variability of the heartbeat with deep breathing or the Valsalva manoeuvre is a good and generally accepted test, although not strongly associated with other PNP test abnormalities. However, axonal degeneration starts in the most distal parts of the axon due to impaired axonal transport. Therefore, the longest C-fibres, i.e. in the lower extremities, are affected first, and incipient changes are most prominent there. For this reason HLDF, a reflex response of the skin blood flow stimulated by heat, has advantages in assessment of early C-fibre dysfunction. Considering the fact that the afferent and efferent sympathetic C-fibres are involved in regulation of microcirculation, the skin blood flow regulation is investigated by means of laser Doppler flowmetry. The microcirculation is stimulated by heat and the reaction of microcirculation is assessed as a value for the function of afferent and efferent (sympathetic) C-fibres. The results of this method are in close correlation with electrophysiologic tests, which is not achieved with sudomotor function.

Autonomic Nervous System↗

PubMed2001

Differential effects of ET-1, ET-2, and ET-3 on pancreatic microcirculation, tissue integrity, and inflammation.

The differential effects of endothelin-1, -2, and -3 (ET-1, ET-2, and ET-3) on pancreatic microcirculation, pancreatic tissue integrity, and an initial inflammatory response, which are three distinct characteristics of acute necrotizing pancreatitis, were investigated in a dose-dependent manner in rats using in vivo microscopy. Red blood cell (RBC) velocity and functional capillary density (FCD) were estimated after topical superfusion of the pancreas with ET-1, ET-2, and ET-3 (100, 10, 1 pmol), revealing that ET-1 (100, 10, 1 pmol) or high ET-2 (100 pmol) and ET-3 (100 pmol) cause a dose-related deterioration of exocrine nutritive pancreatic blood flow. Analysis of pancreatic exocrine tissue damage employing the Spormann score displayed that the ET-mediated microcirculatory impairment was paralleled by dose-dependent tissue damage, which was significant compared to the control group (topical superfusion with 1 ml, saline solution 0.9%). Estimation of pancreatic postcapillary leukocyte accumulation by histomorphologically counting choracetate esterase (CAE) stained leukocytes in 50 high-power fields per animal demonstrated a significant increase in postcapillary accumulation of white blood cells, after topical administration of ET-1, ET-2, and ET-3 compared to controls. In contrast to ET-caused effects on microcirculation and tissue impairment, quantitative analysis of postcapillary leukocyte accumulation revealed the most pronounced effect after ET-2 administration but not after ET-1 administration. This demonstrates that ET-1, ET-2, and ET-3 are all able to mediate microcirculatory impairment, tissue damage, and inflammation. However, ET-3-induced damaging effects are less pronounced, while ET-1 most severely alters microcirculation and ET-2 preferentially induces leukocyte-dependent inflammation.

Animals↗

PubMed1997

Liver metabolic zonation and hepatic microcirculation in carbon tetrachloride-induced experimental cirrhosis.

The exact cause of the hepatic failure in liver cirrhosis is currently unclear, and two main theories have been proposed: the first is based on the altered hepatocyte function (sick hepatocyte hypothesis); the second on the abnormal hepatic architecture (intact hepatocyte hypothesis). Moreover, the microcirculation, a fundamental component in liver structure, shows dramatic changes in cirrhosis that would heavily influence the development of the disease. In order to determine the importance of the microvascular alterations on liver morphofunctional features in experimentally induced cirrhosis, their relationships with structural, ultrastructural, and histoenzymological hepatocyte modifications were investigated. Experimental cirrhosis was induced with controlled intragastric CCl4 administration. Scanning electron microscopy of the vascular corrosion cast technique, associated with light microscopy, transmission electron microscopy, and histoenzymology techniques were employed. The results demonstrated a characteristic micronodular cirrhosis in all the livers studied; the microcirculation displayed the presence of newly formed perinodular plexus. Inside the nodule, areas with two or more hepatocyte-thick laminae were present. Moreover, a rearrangement of the hepatocyte quantitative ultrastructure without real pathological changes and a loss of normal metabolic lobular zonation were noted in the liver parenchyma. These findings support the concept that the progressive modifications of the microcirculation during experimental CC14 cirrhosis modify not only the normal blood flow direction, but also the normal hepatic metabolic gradient with a loss of the normal hepatocytic zonation.

Animals↗

PubMed1991

Three-dimensional reconstruction of the rat brain cortical microcirculation in vivo.

We used confocal laser scanning microscopy (CLSM) to investigate the morphology and three-dimensional relationships of the microcirculation of the superficial layers of the rat brain cortex in vivo. In anesthetized rats equipped with a closed cranial window (dura mater removed), after i.v. injection of 3 mg/100 g of body weight of fluorescein in 0.5 ml of saline, serial optical sections of the brain cortex intraparenchymal microcirculation were taken. Excitation was at a wavelength of 488 nm (argon laser), and emission was collected above 515 nm. CLSM provided images of brain vessels with sufficient signal-to-noise ratio for three-dimensional reconstructions down to a depth of 250 microns beneath the surface of the brain. Compared to conventional fluorescence microscopy, CLSM has a much higher axial resolution and higher depth of penetration. Laser light-induced intravascular aggregates, irregularities of erythrocyte flow, or microvascular occlusions ("light and dye injury") were not apparent in the current experimental paradigm. CLSM is a promising new tool for in vivo visualization of the cerebral microcirculation. Future studies have to characterize the potential damage to the tissue dye mechanisms.

Animals↗

PubMed2004

Diurnal variation in microcirculation of ocular fundus and visual field change in normal-tension glaucoma.

PURPOSE: Diurnal variations in microcirculation of the ocular fundus in normal-tension glaucoma (NTG) were examined to compare with the normal control eyes. The correlation between progression of visual field impairment and diurnal variations in ocular circulation was also studied. METHODS: The subjects were 12 patients with NTG and 12 normal controls. Blood pressure (BP), intraocular pressure (IOP), ocular perfusion pressure (OPP), and square blur rate (SBR), an index of microcirculation acquired by the laser speckle method, were measured at 9 a.m. (morning), 3 p.m. (afternoon), and 9 p.m. (night). Diurnal variations in SBR were tested using the Friedman test and Wilcoxon signed ranks test. On the other hand, diurnal variations in BP, IOP, and OPP were tested by the analysis of variance. The visual field was evaluated at the same time as determining diurnal variation and again about 9 months later to calculate the change. The correlation between variation ratio in SBR and the change in visual field was examined by simple regression. RESULTS: A significant decrease (P=0.04) was found in SBR at night, as compared with the morning value, in the optic nerve head (ONH) of NTG, although no significant diurnal variations were found in SBR either in the choroid-retina or in normal control eyes. Other parameters showed no significant diurnal variations. The larger diurnal variation was in SBR of the ONH, and the more exacerbated visual field impairment was (r=0.59, P=0.04). CONCLUSION: These findings suggest that diurnal variations in the microcirculation of the ONH may play a role in the progression of NTG.

Aged↗

PubMed2006

The Influence of prolonged beta-blockers treatment on male rabbit's sexual behavior and penile microcirculation.

The aim of the study was to assess the effect of the prolonged intake of three beta-blocking drugs (propranolol, metoprolol and nebivolol) on the sexual behavior and penile microcirculation of rabbits. Drugs were administered p.o. for 9 weeks and every three weeks in each group (n=13) one subgroup (n=7) performed behavioral tests, whereas in the second subgroup (n=6) penile microcirculation was measured with a laser Doppler flowmeter. The copulation studies revealed significant impairment of sexual function only in the propranolol treated group. The measured behavioral parameters suggest that at a given dose propranolol affects more performance rather than arousal aspects of rabbits' sexual behavior. In the course of the whole study no significant difference was observed among groups in penile blood flow. The data indicate that among the beta-blockers given only propranolol interferes with sexual behavior, and that beta-blockers do not appear to have a negative effect on penile microcirculation.

Adrenergic beta-Antagonists↗

PubMed2005

Long-term in vivo investigation of mouse cerebral microcirculation by fluorescence confocal microscopy in the area of focal ischemia.

This study was designed to assess that mouse pial and cortical microcirculation can be monitored in the long term directly in the area of focal ischemia, using in vivo fluorescence microscopy. A closed cranial window was placed over the left parieto-occipital cortex of C57BL/6J mice. Local microcirculation was recorded in real time through the window using laser-scanning confocal fluorescence microscopy after intravenous injection of fluorescent erythrocytes and dextran. The basal velocity of erythrocytes through intraparenchymal capillaries was 0.53+/-0.30 mm/sec (n=121 capillaries in 10 mice). Two branches of the middle cerebral artery were topically cauterized through the window. Blood flow evaluated by laser-Doppler flowmetry in two distinct areas indicated the occurrence of an ischemic core (15.2%+/-5.9% of baseline for at least 2 h) and a penumbral zone. Magnetic resonance imaging and histology were used to characterize the ischemic area at 24 h after occlusion. The infarct volume was 7.3+/-3.2 mm(3) (n=6). Microcirculation was repeatedly videorecorded using fluorescence confocal microscopy over the next month. After the decrease following arterial occlusion, capillary erythrocyte velocity was significantly higher than baseline 1 week later, and attained 0.74+/-0.51 mm/sec (n=76 capillaries in six mice, P<0.005) after 1 month, while venous and capillary network remodeling was assessed, with a marked decrease in tortuosity. Immunohistochemistry revealed a zone of necrotic tissue into the infarct epicenter, with activated astrocytes at its border. Such long-term investigations in ischemic cortex brings new insight into the microcirculatory changes induced by focal ischemia and show the feasibility of long-term fluorescence studies in the mouse cortex.

Animals↗

PubMed2006

Modified labeling technique for in vivo visualization of platelets in the cerebral microcirculation of Mongolian gerbils.

Activation of platelets induces interactions with platelets, endothelial cells, and leukocytes. In vivo observation of these interactions in the cerebral microcirculation is rare. The purpose of the present study was to develop a model enabling the in vivo observation of platelet kinetics in the cerebral microcirculation. Intravital fluorescence microscopy was performed in the Mongolian gerbil. Platelets of a donor were labeled ex vivo with carboxyfluorescein diacetat-succinimidylester (CFDA-SE), providing long-term fluorescence. Platelet function was tested ex vivo by flow cytometric analysis and in vivo by analyzing platelet-endothelium interactions. Labeled platelets stimulated with adenosine diphosphate ADP (200 micromol/L) or thrombin (1000 U/L) showed aggregation in flow cytrometric analysis, whereas unstimulated platelets were not aggregated. Irradiation of the brain surface after intravenous injection of the photosensitizing dye Photosan first induced rolling and firm adherence of platelets on arteriolar and venular endothelium, followed by the formation of a thrombus obstructing the vessel. Quantitative analysis (n x 100 microm(-1) min(-1)) before and after 6 mins of irradiation showed 2.6+/-3.2 versus 29.0+/-28.9 rolling, and 0.0+/-0.0 versus 1.7+/-2.3 firm adherent platelets in arterioles, and 3.9+/-3.3 versus 36.6+/-20.9 rolling and 0.0+/-0.0 versus 13.6+/-8.9 firm adherent platelets in venules. Thus, we conclude that ex vivo labeling of platelets with CFDA-SE does not activate platelets. Platelet aggregation and adhesion was achieved by platelet-specific stimulation such as ADP, thrombin or irradiation. In vivo assessment of physiologic and pathophysiologic mechanisms of platelets in the cerebral microcirculation can be achieved in this model.

Adenosine Diphosphate↗

PubMed2000

Quantification of video-taped images in microcirculation research using inexpensive imaging software (Adobe Photoshop).

BACKGROUND: Study end-points in microcirculation research are usually video-taped images rather than numeric computer print-outs. Analysis of these video-taped images for the quantification of microcirculatory parameters usually requires computer-based image analysis systems. Most software programs for image analysis are custom-made, expensive, and limited in their applicability to selected parameters and study end-points. METHODS AND RESULT: We demonstrate herein that an inexpensive, commercially available computer software (Adobe Photoshop), run on a Macintosh G3 computer with inbuilt graphic capture board provides versatile, easy to use tools for the quantification of digitized video images. Using images obtained by intravital fluorescence microscopy from the pre- and postischemic muscle microcirculation in the skinfold chamber model in hamsters, Photoshop allows simple and rapid quantification (i) of microvessel diameters, (ii) of the functional capillary density and (iii) of postischemic leakage of FITC-labeled high molecular weight dextran from postcapillary venules. We present evidence of the technical accuracy of the software tools and of a high degree of interobserver reliability. CONCLUSION: Inexpensive commercially available imaging programs (i.e., Adobe Photoshop) provide versatile tools for image analysis with a wide range of potential applications in microcirculation research.

Animals↗

PubMed2002

Persistence of impaired hepatic microcirculation after nonarterialized liver transplantation in the rat.

OBJECTIVES: The nonarterialized orthotopic rat liver transplant (NOLT) is a frequently used model in transplantation research that was recently used to investigate microcirculatory alterations during acute rejection, which occurs within 7 days. The present study sought to establish whether NOLT represents a reasonable model for the study of the hepatic microcirculation beyond the immediate reperfusion phase. METHODS: Three groups of animals were studied: sham-operated control (n = 8), NOLT (n = 7) and arterialized orthotopic liver transplant (AOLT; n = 8). The hepatic microcirculation was investigated by intravital fluorescence microscopy and laser Doppler flowmetry (LDF) on day 7 postoperatively. Liver histology and plasma levels of liver enzymes were also assessed. RESULTS: Plasma levels of aspartate aminotransferase, alanine aminotransferase, and bilirubin were significantly higher in NOLT than in AOLT and control animals. The low LDF signal recorded from the surface of the NOLT liver (92 +/- 25 vs. 210 +/- 25 and 172 +/- 14 PU in AOLT and control liver, respectively; p < 0.05) was associated with heterogeneous perfusion at both the lobular and sinusoidal levels (density of perfused sinusoids (n/40,000 microm(2)): 5.8 +/- 0.8, 8.1 +/- 0.3, 8.0 +/- 0.3, respectively; p < 0.05). The percentage of hyperfluorescent Ho342-stained hepatocytes (apoptotic) ranged between 2 and 5% and was not significantly different between groups. The lack of post-transplant arterial supply was associated with an increased hepatic cord width-to-sinusoid diameter ratio (3.77 +/- 0.3, 2.02 +/- 0.04, and 1.72 +/- 0.06 in NOLT, AOLT, and control animals, respectively; p < 0.001 vs. control and AOLT) and increased temporary leukocyte adherence to the walls of the terminal hepatic venules. Intense vitamin A autofluorescence around shunt- and large-diameter, slow-velocity vessels was occasionally encountered in the NOLT liver, which coincided with mild fibrosis and bile ductular proliferation. In the well-perfused areas, both AOLT and NOLT were associated with a significant rise in sinusoidal RBC(vel), which was more marked in the NOLT group. CONCLUSIONS: Our data indicate that NOLT represents an inappropriate model for the long-term study of the hepatic microcirculation. Lack of a post-transplant arterial supply may lead to persistent microvascular perfusion failure, hepatocellular/endothelial cell swelling, and microvascular anomalies related to bile duct injury. Recovery from microcirculatory alterations induced by cold preservation/reperfusion injury appears to depend on an intact hepatic arterial blood supply.

Animals↗