Dirk Nolte

Functional Capillary Density: An Indicator of Tissue Perfusion?

Functional capillary density (FCD) is one of the parameters obtained by intravital microscopy using epi-illumination of the tissue surface or trans-illumination of thin tissue layers. FCD, defined as the length of red cell-perfused capillaries per observation area (cm-1), has been used as an indicator of the quality of tissue perfusion in various animal models. Quantitative analysis of FCD in randomly selected regions of the tissue is performed by means of a computer-assisted video analysis system which allows calculation of the length of RBC-perfused capillaries. Basically, two different mathematical approaches can be employed: the first approach is based on the addition of the distances between two neighboring points (pixels) on the video screen (Pythagorean principle). The second approach uses the superimposition of a grid system that allows estimation of the capillary length by counting the number of intersections between the capillaries and the grid lines (stereological approach). The immanent error has been calculated in our laboratory to be +/- 1% with the Pythagorean and +/- 5% with the stereological method. Beside these systematic errors of computerized measurement, the individual (user-dependent) errors occurring during recognition and redrawing of the capillaries on the video image with use of a digitizing tablet are in the range of +/- 10% (intraindividual) and +/- 70% (interindividual) for the recognition and +/- 3% (interindividual) for the redrawing procedure. Our studies indicate that the errors resulting from the use of a computer-assisted calculation (Pythagorean or stereological approach) or the user-assisted redrawing of the capillaries are negligible when compared to the errors made during recognition of the capillaries on the video screen. The methods are applied for assessment of FCD in two different microcirculation models of skin muscle and pancreas yielding highly reproducible, user-independent results under physiologic conditions and the pathophysiologic conditions of ischemia-reperfusion.

Long-term anaesthesia using inhalatory isoflurane in different strains of mice—the haemodynamic effects

The aim of this study was to establish a simple and safe method of anaesthesia for intravital microcirculatory observations in small laboratory animals. The usefulness of isoflurane inhalation anaesthesia has been investigated in different strains of mice commonly used in experimental medicine. These were the hairless (hr/hr, n = 12), the BALB/c (n = 12) and the nude mouse (nu/nu, n = 3). Anaesthesia was maintained by mask inhalation of isoflurane vaporized at concentrations of up to 4% in the induction phase, at 1.5% during acute surgical procedures and at 0.8-1.3% during prolonged experimental observations. Isoflurane was vapoured in a N2O/O2 mixture and saturated with 32-36% FiO2. During observations the body temperature was kept constant at 37°C. The tail artery was cannulated for monitoring of mean arterial blood pressure (MAP) and heart rate (HR). To maintain the body fluid balance, isotonic saline was administered at a constant rate of 0.2 ml/h. Arterial blood samples were drawn for blood-gas analysis at the end of the experiments. All animals survived the anaesthesia protocol lasting between 3 and 6.5 h. During isoflurane inhalation, no breathing complications or changes in systemic circulatory parameters were observed. Mean values of MAP and HR were 79± 3 mmHg and 486± 13 min-1, respectively, over the entire observation period. A moderate acidosis was recorded in animals under isoflurane anaesthesia, with alterations of arterial blood pH, paO2 and pCO2 values (7.29± 0.06, 130± 19 mmHg and 35.6± 4.7 mmHg, respectively). In conclusion, inhalation anaesthesia with isoflurane is useful for experimental studies in the mouse due to (1) the simplicity of administration of the anaesthetic, (2) the rapid induction of anaesthesia, (3) easy control of the depth of anaesthesia, (4) the low percentage of complications, and (5) stable MAP and HR during observations lasting several hours. The proposed technique is especially suitable for observations of the microcirculation under intravital fluorescence microscopy.

Reduction of postischemic leukocyte-endothelium interaction by adenosine via A2 receptor

SummaryThe adhesion of leukocytes to the endothelium of postcapillary venules hallmarks a key event in ischemia-reperfusion injury. Adenosine has been shown to protect from postischemic reperfusion injury, presumably through inhibition of postischemic leukocyte-endothelial interaction. This study was performed to investigate in vivo by which receptors the effect of adenosine on postischemic leukocyte-endothelium interaction is mediated.The hamster dorsal skinfold model and fluorescence microscopy were used for intravital investigation of red cell velocity, vessel diameter, and leukocyte-endothelium interaction in postcapillary venules of a thin striated skin muscle. Leukocytes were stained in vivo with acridine orange (0.5 mg kg−1 min−1 i.v.). Parameters were assessed prior to induction of 4 h ischemia to the muscle tissue and 0.5 h, 2 h, and 24 h after reperfusion. Adenosine, the adenosine A1-selective agonist 2-chloro-N6-cyclopentyladenosine (CCPA), the A2-selective agonist CGS 21,680, the non-selective adenosine receptor antagonist xanthine amine congener (XAC), and the adenosine uptake blocker S-(p-nitrobenzyl)-6-thioinosine (NBTI) were infused via jugular vein starting 15 min prior to release of ischemia until 0.5 h after reperfusion.Adenosine and CGS 21,689 significantly reduced postischemic leukocyte-endothelium interaction 0.5 h after reperfusion (p<0.01), while no inhibitory effect was observed with CCPA. Coadministration of XAC blocked the inhibitory effects of adenosine. Infusion of NBTI alone effectively decreased postischemic leukocyte-endothelium interaction.These findings indicate that adenosine reduces postischemic leukocyte-endothelium interaction via A2 receptor and suggest a protective role of endogenous adenosine during ischemia-reperfusion.

An Improved Intravital Microscopy System

The use of intravital microscopy as a tool for studying the microcirculation has increased greatly over the last several decades. Early microscopes provided the first pictures of the microcirculation, but were cumbersome to use and subjected the tissue to a high light intensity, a problem which has recently become the subject of much discussion. The goal of this project was therefore to build a more ergodynamic microscope which minimizes the light exposure to the tissue. The automation of the microscope controls provides a platform on which other options can be built into the microscope, such as an autofocus feature. Furthermore, the use of the Optimas software also opens the possibility for on-line data processing.

Effects of diaspirin-cross-linked hemoglobin (DCLHbTM) on the microcirculation of striated skin muscle in the hamster: A study on safety and toxicity

Hemoglobin-based oxygen-carrying solutions are reported to exert vasoconstrictor effects and to enhance oxygen radical formation, particularly during ischemia-reperfusion. This study investigates whether diaspirin-cross-linked hemoglobin (DCLHb) affects the microvascular integrity of striated skin muscle. The microcirculation model in the hamster and intravital fluorescence microscopy were applied for investigation of the microvascular changes in striated skin muscle. Hypervolemic infusion (500 mg x kg(-1), I.V.) and isovolemic exchange transfusion (3.3 gm x kg(-1) I.V.; hematocrit 30%) with DCLHb (1) led to a short-lasting (0 to 2 minutes) arteriolar constriction (approximately 20% reduction in baseline diameter), (2) significantly influenced arteriolar vasomotion, (3) increased venular red blood cell velocity by 1.5-fold (p < 0.05 vs dextran, Mr 60,000), and (4) did not enhance microvascular leukocyte-endothelium interaction or endothelial permeability. Resuscitation from severe hemorrhagic shock with autologous blood (AuB) or DCLHb (33 ml x kg(-1), I.V.) immediately restored mean arterial pressure and heart rate, whereas 6% dextran (60 kd)(Dx-60) did not return these parameters to baseline. Venular red blood cell velocity was restored to 110% of baseline after DCLHb, to 90% of baseline after AuB, and to 45% of baseline after Dx-60. Leukocyte-endothelium interaction was significantly enhanced after resuscitation with AuB and Dx-60, whereas this phenomenon was absent after DCLHb. These data demonstrate that DCLHb increases venular red blood cell velocity under both nonischemic and postischemic conditions without inducing enhanced leukocyte-endothelium interaction in the microcirculation of striated skin muscle.

Effects of diaspirin-cross-linked hemoglobin (dclhbtm) on local tissue oxygen tension in striated skin muscle: An efficacy study in the hamster

Using the dorsal skin fold chamber model in the hamster, we analyzed local tissue partial oxygen pressure (PO2) in the striated skin muscle under nonischemic and postischemic conditions with a Clark-type multiwire oxygen surface electrode. Hypervolemic infusion (500 mg x kg(-1) I.V.) or isovolemic exchange transfusion (3.3 gm x kg(-1) I.V.; hematocrit 30%) with diaspirin-cross-linked hemoglobin (DCLHb) resulted in a slight decrease of the mean value of the local tissue PO2 (mm Hg) 1 hour after administration. Concomitantly, the frequency distribution curves of local tissue PO2 values were found to be more narrow (fewer values > 25 mm Hg and < 10 mm Hg). Resuscitation from severe hemorrhagic shock (bleeding of 33 ml x kg(-1) at 0.4 ml x min(-1)) with autologous blood (AuB), Dx-60, or DCLHb led to an increase of mean tissue PO2 values by 4.2-fold (p < 0.05 versus Dx-60), 1.9-fold, and 3.7-fold (p < 0.05 versus Dx-60), respectively, 2 hours after resuscitation. The reduction of tissue hypoxia (0-5 mm Hg) was significant only in the AuB- and DCLHb-treated animals. This study indicates that DCLHb effectively reverses tissue hypoxia after resuscitation from severe hemorrhagic shock by inducing a more homogeneous distribution of the local tissue PO2 levels.

Microcirculatory Models of Ischaemia-Reperf usion in Skin and Striated Muscle

Intravital microscopy is used for analysis of the microcirculation in various organs, e.g. mesentery, intestine, heart, liver and lung, requiring either exteriorization or in situ visualization techniques in anaesthetized animals. In contrast, the implantation of transparent chambers has been employed to allow chronic observation of the microcirculation in intact, non-anaesthetized animals. This paper reports results with two transparent chamber models: the skin-fold chamber model in the hamster and the mouse. An overview is provided of the technical development of the chamber technique and of the various experimental studies that have been performed using these models in ischaemia-reperfusion of striated muscle. Particular emphasis is given to a description of the methods used to induce ischaemia and reperfusion, namely pressure-induced or tourniquet-induced ischaemia. While the former allows simulation of the pathophysiological situation in compartment syndromes, the latter provides an appropriate simulation of the clinical situation in vascular, transplantation and reconstructive surgery. Future possibilities for these microcirculation models in research into the pathophysiology of ischemia-reperfusion are outlined, and potential therapeutic measures to preserve postischaemic tissue are discussed.

Attenuation of leukocyte sequestration by selective blockade of PECAM-1 or VCAM-1 in murine endotoxemia

Background: Molecular mechanisms regulating leukocyte sequestration into the tissue during endotoxemia and/or sepsis are still poorly understood. This in vivo study investigates the biological role of murine PECAM-1 and VCAM-1 for leukocyte sequestration into the lung, liver and striated skin muscle. Methods: Male BALB/c mice were injected intravenously with murine PECAM-1 IgG chimera or monoclonal antibody (mAb) to VCAM-1 (3 mg/kg body weight); controls received equivalent doses of IgG2a (n = 6 per group). Fifteen minutes thereafter, 2 mg/kg body weight of Salmonella abortus equi endotoxin was injected intravenously. At 24 h after the endotoxin challenge, lungs, livers and striated muscle of skin were analyzed for their myeloperoxidase activity. To monitor intravital leukocyte-endothelial cell interactions, fluorescence videomicroscopy was performed in the skin fold chamber model of the BALB/c mouse at 3, 8 and 24 h after injection of endotoxin. Results: Myeloperoxidase activity at 24 h after the endotoxin challenge in lungs (12,171 ± 2,357 mU/g tissue), livers (2,204 ± 238 mU/g) and striated muscle of the skin (1,161 ± 110 mU/g) was significantly reduced in both treatment groups as compared to controls, with strongest attenuation in the PECAM-1 IgG treatment group. Arteriolar leukocyte sticking at 3 h after endotoxin (230 ± 46 cells × mm–2) was significantly reduced in both treatment groups. Leukocyte sticking in postcapillary venules at 8 h after endotoxin (343 ± 69 cells/mm2) was found reduced only in the VCAM-1-mAb-treated animals (215 ± 53 cells/mm2), while it was enhanced in animals treated with PECAM-1 IgG (572 ± 126 cells/mm2). Conclusion: These data show that both PECAM-1 and VCAM-1 are involved in endotoxin-induced leukocyte sequestration in the lung, liver and muscle, presumably through interference with arteriolar and/or venular leukocyte sticking.

Attenuation of postischemic reperfusion injury in striated skin muscle by diaspirin-cross-linked Hb

Hemoglobin-based oxygen carriers have been suggested to enhance the formation of oxygen free radicals, especially under conditions of ischemia-reperfusion (I/R), in which activation and adhesion of leukocytes play a pivotal role for propagation of reperfusion injury. This study investigates the effects of the hemoglobin-based oxygen carrier diaspirin-cross-linked hemoglobin (DCLHb) in an I/R model of hamster striated skin muscle. The dorsal skinfold chamber model in the awake Syrian golden hamster was used for analysis of the microcirculation and local tissue [Formula: see text]in striated skin muscle utilizing the technique of intravital fluorescence microscopy and a multiwire platinum surface (Clark type) electrode. Measurements were made before 4 h of pressure-induced ischemia and at 0.5, 2, and 24 h of reperfusion. Animals were treated with 5 ml/kg body wt of either 10% DCLHb ( n = 8), 6% Dextran 60 (Dx-60; 60 kDa, n = 8), or 0.9% NaCl ( n = 7), which was given intravenously 15 min before reperfusion. In animals treated with DCLHb or Dx-60, a significant decrease of leukocytes rolling along and sticking in postcapillary venules, associated with a recovery of functional capillary density and red blood cell velocity, was observed compared with saline-treated controls. In the early reperfusion period (0.5 h), DCLHb and Dx-60 efficiently restored local tissue[Formula: see text], whereas tissue[Formula: see text] decreased from 18.3 ± 1.9 to 15.3 ± 5.3 mmHg in 0.9% NaCl-treated animals. Electron microscopic analysis of the postischemic tissue at 24 h of reperfusion revealed markedly reduced tissue damage in animals treated with DCLHb compared with Dx-60 or isotonic saline. These results indicate that DCLHb attenuates postischemic reperfusion injury of striated skin muscle, presumably through alterations of leukocyte-endothelial cell interactions.Hemoglobin-based oxygen carriers have been suggested to enhance the formation of oxygen free radicals, especially under conditions of ischemia-reperfusion (I/R), in which activation and adhesion of leukocytes play a pivotal role for propagation of reperfusion injury. This study investigates the effects of the hemoglobin-based oxygen carrier diaspirin-cross-linked hemoglobin (DCLHb) in an I/R model of hamster striated skin muscle. The dorsal skinfold chamber model in the awake Syrian golden hamster was used for analysis of the microcirculation and local tissue PO2 in striated skin muscle utilizing the technique of intravital fluorescence microscopy and a multiwire platinum surface (Clark type) electrode. Measurements were made before 4 h of pressure-induced ischemia and at 0.5, 2, and 24 h of reperfusion. Animals were treated with 5 ml/kg body wt of either 10% DCLHb (n = 8), 6% Dextran 60 (Dx-60; 60 kDa, n = 8), or 0.9% NaCl (n = 7), which was given intravenously 15 min before reperfusion. In animals treated with DCLHb or Dx-60, a significant decrease of leukocytes rolling along and sticking in postcapillary venules, associated with a recovery of functional capillary density and red blood cell velocity, was observed compared with saline-treated controls. In the early reperfusion period (0.5 h), DCLHb and Dx-60 efficiently restored local tissue PO2, whereas tissue PO2 decreased from 18.3 +/- 1.9 to 15.3 +/- 5.3 mmHg in 0.9% NaCl-treated animals. Electron microscopic analysis of the postischemic tissue at 24 h of reperfusion revealed markedly reduced tissue damage in animals treated with DCLHb compared with Dx-60 or isotonic saline. These results indicate that DCLHb attenuates postischemic reperfusion injury of striated skin muscle, presumably through alterations of leukocyte-endothelial cell interactions.

Compatibility of different colloid plasma expanders with perflubron emulsion : An intravital microscopic study in the hamster

BackgroundPerfluorocarbon-based oxygen carriers have been proposed as an adjunct to autologous blood conservation techniques during elective surgery. To date, the effects of perfluorocarbon emulsions at the microcirculatory level have not been studied extensively. In this study the effects of perflubron emulsion on the microcirculation after acute normovolemic hemodilution (ANH) were investigated using different colloid plasma expanders. MethodsThe dorsal skin fold chamber model and intravital fluorescence microscopy were used for analysis of the microcirculation in the thin striated skin muscle of conscious hamsters (body weight, 40–60 g). Measurements of microvascular perfusion and leukocyte adhesion (n = 6 animals per experimental group) were made before and at 10, 30, and 60 min after ANH (to hematocrit 0.3) with either 6% hydroxyethyl starch 200/0.6 (HES), 3.5% gelatin, 5% human serum albumin (HSA), or 6% dextran 60 (DX-60) followed by intravenous injection of 3 ml/kg body weight of a 60% weight/volume perfluorocarbon emulsion based on perflubron (perfluorooctyl bromide) emulsified with egg yolk lecithin. ResultsAcute normovolemic hemodilution with HES, gelatin, or HSA followed by injection of perflubron emulsion elicited no alterations of local microvascular perfusion or leukocyte–endothelium interaction as assessed in arterioles and postcapillary venules. However, ANH with DX-60 followed by injection of perflubron emulsion led to a significant reduction of erythrocyte velocity in postcapillary venules and an increase in venular leukocyte sticking that was never observed with DX-60 alone. ConclusionsHydroxyethyl starch, gelatin, and HSA are compatible with perflubron emulsion in the setting of ANH. Only DX-60 appeared to be incompatible with perflubron emulsion, as evidenced by impairment of capillary perfusion.